Assays for TIMP2 having improved performance in biological samples

ABSTRACT

The present invention relates to antibodies or antigen binding fragments thereof that binds to human “TIMP2. Further provided are methods for treating subjects, including human subjects, in need of treatment with the isolated TIMP2 antibodies or antigen-binding fragments thereof disclosed herein. Further provided are pharmaceutical or sterile compositions of anti-TIMP2 antibodies and antigen-binding fragments of the invention, the antibody or antigen-binding fragment thereof is admixed with a pharmaceutically acceptable carrier or excipient. Further provided are kits comprising one or more components that include an anti-TIMP2 antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition thereof.

The present application claims the benefit of U.S. Provisional PatentApplication No. 62/482,089 filed Apr. 5, 2017, which is herebyincorporated by reference in its entirety, including all tables,figures, and claims.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted inASCII format via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Oct. 11, 2018, is namedASTM_005_PCT_SeqListing.txt and is 29,778 bytes in size.

BACKGROUND

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

Metalloproteinase inhibitor 2 (Swiss-Prot P16035, also known as “Tissueinhibitor of metalloproteinases 2” and “TIMP2”) is a secreted proteinwhich complexes with metalloproteinases and irreversibly inactivatesthem by binding to their catalytic zinc cofactor. TIMP2 is known to acton MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-13, MMP-14,MMP-15, MMP-16 and MMP-19. TIMP2 reportedly suppresses the proliferationof endothelial cells. As a result, the encoded protein has beensuggested to have a role in the maintenance of tissue homeostasis bysuppressing the proliferation of quiescent tissues in response toangiogenic factors, and by inhibiting protease activity in tissuesundergoing remodeling.

In addition, WO2010/048346 and WO2011/075744, each of which is herebyincorporated by reference in its entirety including all tables, figuresand claims, describe the use of TIMP2 for evaluating the renal status ofa subject both individually and in multimarker panels. In particular,TIMP2 levels measured by immunoassay are shown to correlate to riskstratification, diagnosis, staging, prognosis, classifying andmonitoring of the renal status. PCT/US2014/050195 discloses certainantibodies for use in such assays.

Signals obtained from specific binding assays such as immunoassays are adirect result of complexes formed between one or more binding species(e.g., antibodies) and the target biomolecule (i.e., the analyte) andpolypeptides containing the necessary epitope(s) to which the antibodiesbind. Immunoassays are often able to “detect” an analyte; but because anantibody epitope is on the order of 8 amino acids, an immunoassayconfigured to detect a marker of interest will also detect polypeptidesrelated to the marker sequence, so long as those polypeptides containthe epitope(s) necessary to bind to the antibody or antibodies used inthe assay. While such assays may detect the full length biomarker andthe assay result be expressed as a concentration of a biomarker ofinterest, the signal from the assay is actually a result of all such“immunoreactive” polypeptides present in the sample. Such binding assaysmay also detect immunoreactive polypeptides present in a biologicalsample that are complexed to additional species, such as bindingproteins, receptors, heparin, lipids, sugars, etc., provided that thoseadditional species do not interfere in binding between the bindingspecies and the target biomolecule. Typically, however, specific bindingassays are formulated using purified analyte, and complex formation andfragmentation patterns are not considered. This is particularly truewhere the identity of such additional binding species are unknown.

SUMMARY

It is an object of the invention to provide antibodies which bind novelepitopes of TIMP2. Such antibodies can find use in immunoassays withimproved clinical performance, particularly when used in the evaluationof renal injuries, and in therapeutic methods in which TIMP2 binding isdesired.

In a first aspect, the present invention relates to antibodies orantigen binding fragments thereof that binds to human TIMP2, wherein theantibody or antigen binding fragment comprises one or more, andoptionally each, of:

-   -   a light chain variable region CDR1 comprising the amino acid        sequence ESISGW (SEQ ID NO:1) or an amino acid sequence at least        90%, 95%, 97%, 98%, or 99% identical thereto,    -   a light chain variable region CDR2 comprising the amino acid        sequence RAS (SEQ ID NO:2) or an amino acid sequence at least        90%, 95%, 97%, 98%, or 99% identical thereto,    -   a light chain variable region CDR3 comprising the amino acid        sequence QCSYGINGNSEHGNP (SEQ ID NO:3) or an amino acid sequence        at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a heavy chain variable region CDR1 comprising the amino acid        sequence GFTISSNYY (SEQ ID NO:4) or an amino acid sequence at        least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a heavy chain variable region CDR2 comprising the amino acid        sequence ILGGSGTYT (SEQ ID NO:5) or an amino acid sequence at        least 90%, 95%, 97%, 98%, or 99% identical thereto, and    -   a heavy chain variable region CDR3 comprising the amino acid        sequence ARQAPADTYLYGGYGPFNL (SEQ ID NO:6) or an amino acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

In certain embodiments, such antibodies or antigen binding fragmentscomprise

-   -   a light chain sequence comprising the amino acid sequence of SEQ        ID NO:1 or an amino acid sequence at least 90%, 95%, 97%, 98%,        or 99% identical thereto; the amino acid sequence of SEQ ID NO:2        or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%        identical thereto; and/or the amino acid sequence of SEQ ID NO:3        or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%        identical thereto; and/or    -   a heavy chain sequence comprising the amino acid sequence of SEQ        ID NO:4 or an amino acid sequence at least 90%, 95%, 97%, 98%,        or 99% identical thereto; the amino acid sequence of SEQ ID NO:5        or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%        identical thereto; and/or the amino acid sequence of SEQ ID NO:6        or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%        identical thereto.

In certain preferred embodiments, such antibodies or antigen bindingfragments comprise

-   -   a light chain variable region sequence comprising the amino acid        sequence DIVMTQTPSSVEAAVGGTVTIKCQASESISGWLAWYQQKPGQPPKLLIYR        ASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQCSYGINGNSEHG NPFGGGTEVVVK        (SEQ ID NO: 7) or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% identical thereto; and/or    -   a heavy chain variable region sequence comprising the amino acid        sequence QSLEESGGDLVKPEGSLTLTCTASGFTISSNYYMCWVRQAPGKGLEWVA        CILGGSGTYTYYATWAKGRFTISKTSSTTVTLQMPSLTAADTATYFCARQ        APADTYLYGGYGPFNLWGQGTLVTVSS (SEQ ID NO: 8) or an amino acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

In certain most preferred embodiments, such antibodies or antigenbinding fragments comprise a heavy chain variable region sequencecomprising the amino acid sequence of SEQ ID NO: 8 and a light chainvariable region sequence comprising the amino acid sequence of SEQ IDNO: 7.

In certain most preferred embodiments, such antibodies or antigenbinding fragments comprise a heavy chain sequence comprising the aminoacid sequence of SEQ ID NO: 10 and a light chain sequence comprising theamino acid sequence of SEQ ID NO:16. Such antibodies are referred toherein as antibody “40H2-40K3.”

In still another related aspect, the antibodies or antigen bindingfragments described above are a member of a pair of antibodies whichform a sandwich complex with a second antibody or antigen bindingfragment that also binds to TIMP2. By way of example only, the secondantibody may be an antibody described below as 6E2.1 or an antigenbinding fragment thereof.

In a related aspect, the present invention relates to antibodies orbinding fragments which bind to TIMP2, for example antibody 40H2-40K3,wherein the antibody binds to the sequence NHRYQMGCECKI (SEQ ID NO: 21).Preferred antibodies which bind to this sequence are the antibodiesdescribed above, and most preferably such antibodies or antigen bindingfragments comprise a heavy chain variable region sequence comprising theamino acid sequence of SEQ ID NO: 8 or an amino acid sequence at least90%, 95%, 97%, 98%, or 99% identical thereto and a light chain variableregion sequence comprising the amino acid sequence of SEQ ID NO: 7 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto. In certain most preferred embodiments, such antibodies orantigen binding fragments comprise a heavy chain variable regionsequence comprising the amino acid sequence of SEQ ID NO: 8 and a lightchain variable region sequence comprising the amino acid sequence of SEQID NO: 7. Epitope binding is preferably determined by alanine scanningusing non-mutated (peptide set 1) and double alanine mutations (peptideset 2) within a nested set of linear 15-mer peptides with a stepping ofone amino acid residue derived from the TIMP-2 sequence (SEQ ID NO: 45).See, e.g., McBride et al., Methods Mol. Biol. 1352: 67-83, 2016; Kong etal., J. Virol. 86: 12115-28, 2012.

In certain embodiments, the antibody that binds to the sequenceNHRYQMGCECKI (SEQ ID NO: 21) binds to a discontinuous epitope. In theseembodiments, the antibodies or binding fragments which bind to TIMP2further bind to the sequence HPQQAFCNA (SEQ ID NO: 22) and to thesequence RSDGSCAWYR (SEQ ID NO: 23).

In a related aspect, the present invention relates to nucleic acidsencoding such antibodies or antigen binding fragments thereof that bindsto human TIMP2.

In another aspect, the present invention relates to antibodies orantigen binding fragments thereof that binds to human TIMP2, wherein theantibody or antigen binding fragment comprises one or more, andoptionally each, of:

-   -   a light chain variable region CDR1 comprising the amino acid        sequence DHINNW (SEQ ID NO: 24) or an amino acid sequence at        least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a light chain variable region CDR2 comprising the amino acid        sequence SGA (SEQ ID NO: 25) or an amino acid sequence at least        90%, 95%, 97%, 98%, or 99% identical thereto,    -   a light chain variable region CDR3 comprising the amino acid        sequence QQYWSTPFT (SEQ ID NO: 26) or an amino acid sequence at        least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a heavy chain variable region CDR1 comprising the amino acid        sequence GYSFTSYW (SEQ ID NO: 27) or an amino acid sequence at        least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a heavy chain variable region CDR2 comprising the amino acid        sequence IDPSDSET (SEQ ID NO: 28) or an amino acid sequence at        least 90%, 95%, 97%, 98%, or 99% identical thereto, and    -   a heavy chain variable region CDR3 comprising the amino acid        sequence ARRDYGSRYDAMDY (SEQ ID NO: 29) or an amino acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

In certain embodiments, such antibodies or antigen binding fragmentscomprise

-   -   a light chain sequence comprising the amino acid sequence of SEQ        ID NO: 24 or an amino acid sequence at least 90%, 95%, 97%, 98%,        or 99% identical thereto; the amino acid sequence of SEQ ID NO:        25 or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%        identical thereto; and/or the amino acid sequence of SEQ ID NO:        26 or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%        identical thereto; and/or    -   a heavy chain sequence comprising the amino acid sequence of SEQ        ID NO: 27 or an amino acid sequence at least 90%, 95%, 97%, 98%,        or 99% identical thereto; the amino acid sequence of SEQ ID NO:        28 or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%        identical thereto; and/or the amino acid sequence of SEQ ID NO:        29 or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%        identical thereto.

In certain preferred embodiments, such antibodies or antigen bindingfragments comprise

-   -   a light chain variable region sequence comprising the amino acid        sequence DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISG        ATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPFTFGSG TKLEIK (SEQ        ID NO: 30) or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% identical thereto; and/or    -   a heavy chain variable region sequence comprising the amino acid        sequence QVQLQQSGPQLVRPGASVKISCKASGYSFTSYWMHWVKQRPGQGLEWIG VI    -   DPSDSETRLNQKFKDKATLTVDKSSSTAYMQLNSPTSEDSAVYYCARRD Y        GSRYDAMDYWGQGTSVTVSS (SEQ ID NO: 31) or an amino acid sequence        at least 90%, 95%, 97%, 98%, or 99% identical thereto.

In certain most preferred embodiments, such antibodies or antigenbinding fragments comprise a heavy chain variable region sequencecomprising the amino acid sequence of SEQ ID NO: 31 and a light chainvariable region sequence comprising the amino acid sequence of SEQ IDNO: 30.

In certain most preferred embodiments, such antibodies or antigenbinding fragments comprise a heavy chain sequence comprising the aminoacid sequence of SEQ ID NO: 33 and a light chain sequence comprising theamino acid sequence of SEQ ID NO:39. Such antibodies are referred toherein as antibody “6E2.1.”

In still another related aspect, the antibodies or antigen bindingfragments described above are a member of a pair of antibodies whichform a sandwich complex with a second antibody or antigen bindingfragment that also binds to TIMP2. By way of example only, the secondantibody may be an antibody described above as 40H2-40K3 or an antigenbinding fragment thereof.

In a related aspect, the present invention relates to antibodies orbinding fragments which bind to TIMP2, for example antibody 6E2.1,wherein the antibody binds to the sequence PWDTLSTTQKK (SEQ ID NO: 44).Preferred antibodies which bind to this sequence are the antibodiesdescribed above, and most preferably such antibodies or antigen bindingfragments comprise a heavy chain variable region sequence comprising theamino acid sequence of SEQ ID NO: 31 or an amino acid sequence at least90%, 95%, 97%, 98%, or 99% identical thereto and a light chain variableregion sequence comprising the amino acid sequence of SEQ ID NO:30 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto. In certain most preferred embodiments, such antibodies orantigen binding fragments comprise a heavy chain variable regionsequence comprising the amino acid sequence of SEQ ID NO: 31 and a lightchain variable region sequence comprising the amino acid sequence of SEQID NO: 30. Epitope binding is preferably determined by alanine scanningusing single and double alanine mutations within a nested set of linear15-mer peptides with a stepping of one amino acid residue derived fromthe TIMP-2 sequence (SEQ ID NO: 45). See, e.g., McBride et al., MethodsMol. Biol. 1352: 67-83, 2016; Kong et al., J. Virol. 86: 12115-28, 2012.

In a related aspect, the present invention relates to nucleic acidsencoding such antibodies or antigen binding fragments thereof that bindsto human TIMP2.

Antibodies or antigen binding fragments for use in the claimed methodsmay be obtained from a variety of species. For example, the antibodiesor antigen binding fragments of the present invention may compriseimmunoglobulin sequences which are rabbit, mouse, rat, guinea pig,chicken, goat, sheep, donkey, human, llama or camelid sequences, orcombinations of such sequences (so-called chimeric antibodies). Suchantibodies or antigen binding fragments may also be monoclonal orpolyclonal. Antibodies for use in the present invention may beidentified by their performance in immunoassays, and then furthercharacterized by epitope mapping in order to understand the epitopeswhich are relevant to that performance. Preferred are rabbit antibodiesor humanized versions derived from rabbit antibodies.

Such antibodies or antigen binding fragments may be conjugated to asignal development element or immobilized on a solid support. Inaddition, such antibodies or antigen binding fragments may be used in anumber of competitive and sandwich assay formats. In an example of asandwich assay, a first antibody or antigen binding fragment (detectablylabeled) and a second antibody or antigen binding fragment (immobilizedat a predetermined zone of a test device) form sandwich complexes withTIMP2 in the sample at a predetermined zone of a test device. Insandwich assays, the first and second antibodies or antigen bindingfragments can be the same (particularly when polyclonal antibodies areused) or different. Thus, the antibodies or antigen binding fragments ofthe invention may be used in sandwich pairs or may be used individuallywith another binding entity which is not a monoclonal antibody such as apolyclonal antibody or an aptamer.

The antibodies or antigen binding fragments of the present invention canbe used as reagents in test kits for detecting TIMP-2 in biologicalsamples. Such a test kit may, for example, comprise a disposable testdevice configured to generate a detectable signal related to the presentor amount of human TIMP-2 in a biological sample. Alternatively, such atest kit may be formulated for performing an assay in a clinicalanalyzer which does not utilize a disposable test device. Preferably,the test kit is an in vitro diagnostic. The term “in vitro diagnostic”as used herein refers to a medical device which is a reagent, reagentproduct, calibrator, control material, kit, instrument, apparatus,equipment, or system, whether used alone or in combination, intended bythe manufacturer to be used in vitro for the examination of specimens,including blood and tissue donations, derived from the human body,solely or principally for the purpose of providing informationconcerning a physiological or pathological state, or concerning acongenital abnormality, or to determine the safety and compatibilitywith potential recipients, or to monitor therapeutic measures.

In certain embodiments, the immunoassay is performed in a lateral flowformat. Lateral flow tests are a form of immunoassay in which the testsample flows in a chromatographic fashion along a bibulous ornon-bibulous porous solid substrate. Lateral flow tests can operate aseither competitive or sandwich format assays. Preferred lateral flowdevices are disposable, single use test devices. A sample is applied tothe test device at an application zone and transits the substrate, whereit encounters lines or zones which have been pretreated with an antibodyor antigen. The term “test zone” as used herein refers to a discretelocation on a lateral flow test strip which is interrogated in order togenerate a signal related to the presence or amount of an analyte ofinterest. The detectable signal may be read visually or obtained byinserting the disposable test device into an analytical instrument suchas a reflectometer, a fluorometer, or a transmission photometer. Thislist is not meant to be limiting. Sample may be applied withoutpretreatment to the application zone, or may be premixed with one ormore assay reagents prior to application. In the latter case, theantibody may be provided in a separate container from the disposabletest device.

An antibody or antigen binding fragment of the present invention may bediffusively immobilized to a surface within a disposable test device,such that the antibody or antigen binding fragment dissolves into asample when the sample contacts the surface. In a sandwich assay format,this diffusively bound antibody may bind to its cognate antigen in thesample, and then be immobilized at a detection zone when the antigen isbound by a second antibody or antigen binding fragment non-diffusivelybound at the detection zone. In a competitive format, its cognateantigen in the sample may compete for binding to the diffusively boundantibody with a labeled antigen provided as an assay reagent.

A kit of the invention can further comprise a calibration to relate thedetectable signal to a concentration of TIMP-2. By way of example, acalibration curve may be provided on an electronic memory device whichis read by the analytical instrument which receives the disposable testdevice, such as a ROM chip, a flash drive, an RFID tag, etc.Alternatively, the calibration curve may be provided on an encoded labelwhich is read optically, such as a 2-D bar code, or transmitted via anetwork connection. The analytical instrument can then use thiscalibration curve to relate a detectable signal from an assay into aTIMP-2 concentration

In certain embodiment, an assay method performed using one or moreantibodies of the present invention provides a signal related to thepresent or amount of human TIMP-2 in a biological sample, wherein theminimum detectable concentration of TIMP-2 in the assay method is 10ng/mL or less, more preferably 1 ng/mL or less, and most preferably 0.1ng/mL or less.

In related aspects, the present invention provides methods fordetermining the presence or amount of human TIMP2 in a biologicalsample, comprising:

performing an immunoassay on the biological sample with a firstmonoclonal antibody or antigen binding fragment and a second monoclonalantibody or antigen binding fragment which together form sandwichcomplexes with human TIMP2, wherein the immunoassay provides adetectable signal that is related to the presence or amount of humanTIMP2 in the biological sample bound in the sandwich complexes; andrelating the detectable signal to the presence or amount of human TIMP2in the biological sample. Preferably, the minimum detectableconcentration of TIMP-2 in the immunoassay is 10 ng/mL or less, morepreferably 1 ng/mL or less, and most preferably 0.1 ng/mL or less.

In particularly preferred embodiments, one or both of the firstmonoclonal antibody or antigen binding fragment and second monoclonalantibody or antigen binding fragment is an antibody of the presentinvention as described herein. In certain embodiments,

the first monoclonal antibody or antigen binding fragment comprises aheavy chain variable region sequence comprising the amino acid sequenceof SEQ ID NO: 8 or an amino acid sequence at least 90%, 95%, 97%, 98%,or 99% identical thereto and a light chain variable region sequencecomprising the amino acid sequence of SEQ ID NO: 7 or an amino acidsequence at least 90%, 95%, 97%, 98%, or 99% identical thereto, andthe second monoclonal antibody or antigen binding fragment comprises aheavy chain variable region sequence comprising the amino acid sequenceof SEQ ID NO: 31 or an amino acid sequence at least 90%, 95%, 97%, 98%,or 99% identical thereto and a light chain variable region sequencecomprising the amino acid sequence of SEQ ID NO: 30 or an amino acidsequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

In particularly preferred embodiments, the first monoclonal antibody orantigen binding fragment is 40H2-40K3 or antigen binding fragmentthereof and the second monoclonal antibody or antigen binding fragmentis 6E2.1 or antigen binding fragment thereof.

Preferred assay methods comprise performing an immunoassay that detectshuman TIMP-2. Such immunoassays may comprise contacting said body fluidsample with an antibody or antigen binding fragment that detects themarker and detecting binding to that antibody. While the presentinvention is generally described in terms of immunoassays, other bindingentities (e.g., aptamers) which are not based on an immunoglobulinscaffold may be used in lieu of antibodies in such methods. Preferably,the body fluid sample is selected from the group consisting of urine,saliva, blood, serum, and plasma, and most preferably urine.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages of the disclosure will be apparent from thedescription and drawings, and from the claims.

DETAILED DESCRIPTION

Definitions

As used herein, the terms “Metalloproteinase inhibitor 2” and “TIMP-2”refer to one or more polypeptides present in a biological sample thatare derived from the Metalloproteinase inhibitor 2 precursor (humanprecursor: Swiss-Prot P16035 (SEQ ID NO: 45)).

        10         20         30         40        50          60MGAAARTLRL ALGLLLLATL LRPADACSCS PVHPQQAFCN ADVVIRAKAV SEKEVDSGND        70         80         90        100        110        120IYGNPIKRIQ YEIKQIKMFK GPEKDIEFIY TAPSSAVCGV SLDVGGKKEY LIAGKAEGDG       130        140        150        160        170        180KMHITLCDFI VPWDTLSTTQ KKSLNHRYQM GCECKITRCP MIPCYISSPD ECLWMDWVTE       190        200        210        220KNINGHQAKF FACIKRSDGS CAWYRGAAPP KQEFLDIEDP

The following domains have been identified in Metalloproteinaseinhibitor 2:

Residues Length Domain ID  1-26 26 Signal peptide 27-220 194Metalloproteinase inhibitor 2

Unless specifically noted otherwise herein, the definitions of the termsused are standard definitions used in the art of pharmaceuticalsciences. As used in the specification and the appended claims, thesingular forms “a,” “an” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “apharmaceutical carrier” includes mixtures of two or more such carriers,and the like.

The term “subject” as used herein refers to a human or non-humanorganism. Thus, the methods and compositions described herein areapplicable to both human and veterinary disease. Further, while asubject is preferably a living organism, the invention described hereinmay be used in post-mortem analysis as well. Preferred subjects arehumans, and most preferably “patients,” which as used herein refers toliving humans that are receiving medical care for a disease orcondition. This includes persons with no defined illness who are beinginvestigated for signs of pathology.

Preferably, an analyte is measured in a sample. Such a sample may beobtained from a subject, or may be obtained from biological materialsintended to be provided to the subject. For example, a sample may beobtained from a kidney being evaluated for possible transplantation intoa subject, and an analyte measurement used to evaluate the kidney forpreexisting damage. Preferred samples are body fluid samples.

The term “body fluid sample” as used herein refers to a sample of bodilyfluid obtained for the purpose of diagnosis, prognosis, classificationor evaluation of a subject of interest, such as a patient or transplantdonor. In certain embodiments, such a sample may be obtained for thepurpose of determining the outcome of an ongoing condition or the effectof a treatment regimen on a condition. Preferred body fluid samplesinclude blood, serum, plasma, cerebrospinal fluid, urine, saliva,sputum, and pleural effusions. In addition, one of skill in the artwould realize that certain body fluid samples would be more readilyanalyzed following a fractionation or purification procedure, forexample, separation of whole blood into serum or plasma components.

The term “diagnosis” as used herein refers to methods by which theskilled artisan can estimate and/or determine the probability (“alikelihood”) of whether or not a patient is suffering from a givendisease or condition. In the case of the present invention, “diagnosis”includes using the results of an assay, most preferably an immunoassay,for a kidney injury marker of the present invention, optionally togetherwith other clinical characteristics, to arrive at a diagnosis (that is,the occurrence or nonoccurrence) of an acute renal injury or ARF for thesubject from which a sample was obtained and assayed. That such adiagnosis is “determined” is not meant to imply that the diagnosis is100% accurate. Many biomarkers are indicative of multiple conditions.The skilled clinician does not use biomarker results in an informationalvacuum, but rather test results are used together with other clinicalindicia to arrive at a diagnosis. Thus, a measured biomarker level onone side of a predetermined diagnostic threshold indicates a greaterlikelihood of the occurrence of disease in the subject relative to ameasured level on the other side of the predetermined diagnosticthreshold.

Similarly, a prognostic risk signals a probability (“a likelihood”) thata given course or outcome will occur. A level or a change in level of aprognostic indicator, which in turn is associated with an increasedprobability of morbidity (e.g., worsening renal function, future ARF, ordeath) is referred to as being “indicative of an increased likelihood”of an adverse outcome in a patient.

The term “lateral flow” as used herein refers to flow of reagents in alongitudinal direction through a substantially flat porous material.Such porous material is “substantially flat” if the thickness of thematerial is no more than 10% of the length and width dimensions.

The term “downstream region” as used herein relative to a first regionof a device refers to which receives fluid flow after that fluid hasalready reached the first region.

The term “sample application region” as used herein refers to a portionof an assay device into which a fluid sample of interest is introducedfor purposes of determining a component thereof.

Marker Assays

In general, immunoassays involve contacting a sample containing orsuspected of containing a biomarker of interest with at least oneantibody that specifically binds to the biomarker. A signal is thengenerated indicative of the presence or amount of complexes formed bythe binding of polypeptides in the sample to the antibody. The signal isthen related to the presence or amount of the biomarker in the sample.Numerous methods and devices are well known to the skilled artisan forthe detection and analysis of biomarkers. See, e.g., U.S. Pat. Nos.6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272;5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and5,480,792, and The Immunoassay Handbook, David Wild, ed. Stockton Press,New York, 1994, each of which is hereby incorporated by reference in itsentirety, including all tables, figures and claims.

The assay devices and methods known in the art can utilize labeledmolecules in various sandwich, competitive, or non-competitive assayformats, to generate a signal that is related to the presence or amountof the biomarker of interest. Suitable assay formats also includechromatographic, mass spectrographic, and protein “blotting” methods.Additionally, certain methods and devices, such as biosensors andoptical immunoassays, may be employed to determine the presence oramount of analytes without the need for a labeled molecule. See, e.g.,U.S. Pat. Nos. 5,631,171; and 5,955,377, each of which is herebyincorporated by reference in its entirety, including all tables, figuresand claims. One skilled in the art also recognizes that roboticinstrumentation including but not limited to Beckman ACCESS®, AbbottAXSYM®, Roche ELECSYS®, Dade Behring STRATUS® systems are among theimmunoassay analyzers that are capable of performing immunoassays. Butany suitable immunoassay may be utilized, for example, enzyme-linkedimmunoassays (ELISA), radioimmunoassays (RIAs), competitive bindingassays, and the like.

Antibodies or other polypeptides may be immobilized onto a variety ofsolid supports for use in assays. Solid phases that may be used toimmobilize specific binding members include those developed and/or usedas solid phases in solid phase binding assays. Examples of suitablesolid phases include membrane filters, cellulose-based papers, beads(including polymeric, latex and paramagnetic particles), glass, siliconwafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels,SPOCC gels, and multiple-well plates. An assay strip could be preparedby coating the antibody or a plurality of antibodies in an array onsolid support. This strip could then be dipped into the test sample andthen processed quickly through washes and detection steps to generate ameasurable signal, such as a colored spot. Antibodies or otherpolypeptides may be bound to specific zones of assay devices either byconjugating directly to an assay device surface, or by indirect binding.In an example of the later case, antibodies or other polypeptides may beimmobilized on particles or other solid supports, and that solid supportimmobilized to the device surface.

Biological assays require methods for detection, and one of the mostcommon methods for quantitation of results is to conjugate a detectablelabel to a protein or nucleic acid that has affinity for one of thecomponents in the biological system being studied. Detectable labels mayinclude molecules that are themselves detectable (e.g., fluorescentmoieties, electrochemical labels, metal chelates, etc.) as well asmolecules that may be indirectly detected by production of a detectablereaction product (e.g., enzymes such as horseradish peroxidase, alkalinephosphatase, etc.) or by a specific binding molecule which itself may bedetectable (e.g., biotin, digoxigenin, maltose, oligohistidine,2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

Preparation of solid phases and detectable label conjugates oftencomprise the use of chemical cross-linkers. Cross-linking reagentscontain at least two reactive groups, and are divided generally intohomofunctional cross-linkers (containing identical reactive groups) andheterofunctional cross-linkers (containing non-identical reactivegroups). Homobifunctional cross-linkers that couple through amines,sulfhydryls or react nonspecifically are available from many commercialsources. Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyldisulfides are thiol reactive groups. Maleimides, alkyl and arylhalides, and alpha-haloacyls react with sulfhydryls to form thiol etherbonds, while pyridyl disulfides react with sulfhydryls to produce mixeddisulfides. The pyridyl disulfide product is cleavable. Imidoesters arealso very useful for protein-protein cross-links. A variety ofheterobifunctional cross-linkers, each combining different attributesfor successful conjugation, are commercially available.

In certain aspects, the present invention provides kits for the analysisof the described marker. The kit comprises reagents for the analysis ofat least one test sample which comprise at least one antibody thatspecifically binds to the marker. The kit can also include devices andinstructions for performing one or more of the diagnostic and/orprognostic correlations described herein. Preferred kits will comprisean antibody pair for performing a sandwich assay, or a labeled speciesfor performing a competitive assay, for the analyte. Preferably, anantibody pair comprises a first antibody conjugated to a solid phase anda second antibody conjugated to a detectable label, wherein each of thefirst and second antibodies that bind a kidney injury marker. Mostpreferably each of the antibodies are monoclonal antibodies. Theinstructions for use of the kit and performing the correlations can bein the form of labeling, which refers to any written or recordedmaterial that is attached to, or otherwise accompanies a kit at any timeduring its manufacture, transport, sale or use. For example, the termlabeling encompasses advertising leaflets and brochures, packagingmaterials, instructions, audio or video cassettes, computer discs, aswell as writing imprinted directly on kits.

In certain embodiments, the marker assay is performed using a single-usedisposable test device. Such test devices often take the form of lateralflow devices which are now familiar from the common use ofover-the-counter pregnancy tests. Generally, these assay devices have anextended base layer on which a differentiation can be made between asample addition region and an evaluation region. In typical use, thesample is applied to the sample addition region, flows along a liquidtransport path which runs parallel to the base layer, and then flowsinto the evaluation region. A capture reagent is present in theevaluation region, and the captured analyte can be detected by a varietyof protocols to detect visible moieties associated with the capturedanalyte. For example, the assay may produce a visual signal, such ascolor change, fluorescence, luminescence, and the like, when indicatingthe presence or absence of an analyte in a biological sample.

A sample addition region can be provided, for example, in the form of anopen chamber in a housing; in the form of an absorbent pad; etc. Thesample addition region can be a port of various configurations, that is,round, oblong, square and the like or the region can be a trough in thedevice.

A filter element can be placed in, on, or adjacent to the sampleaddition region to filter particulates from the sample, such as toremove or retard blood cells from blood so that plasma can furthertravel through the device. Filtrate can then move into a porous memberfluidly connected to the filter. Suitable filters for removing orretarding cellular material present in blood are well known in the art.See, e.g., U.S. Pat. Nos. 4,477,575; 5,166,051; 6,391,265; and7,125,493, each of which is hereby incorporated by reference in itsentirety. Many suitable materials are known to skilled artisans, and caninclude glass fibers, synthetic resin fibers, membranes of various typesincluding asymmetric membrane filters in which the pore size varies fromabout 65 to about 15 μm, and combinations of such materials. Inaddition, a filter element can comprise one or more chemical substancesto facilitate separation of red blood cells from blood plasma. Examplesof such chemical substances are thrombin, lectins, cationic polymers,antibodies against one or more red blood cell surface antigens and thelike. Such chemical substance(s) which facilitate separation of redblood cells from plasma may be provided in the filter element bycovalent means, nonspecific absorption, etc.

In certain embodiments, a label zone is located downstream of the samplereceiving zone, and contains a diffusively located labeled reagent thatbinds to the analyte of interest or that competes with the analyte ofinterest for binding to a binding species. Alternatively, the label zonecan be eliminated if the labeled reagent is premixed with the sampleprior to application to the sample receiving zone. A detection zone isdisposed downstream of from the label zone, and contains an immobilizedcapture reagent that binds to the analyte of interest.

The optimum pore diameter for the membrane for use in the invention isabout 10 to about 50 μm. The membranes typically are from about 1 mil toabout 15 mils in thickness, typically in the range of from 5 or 10 mils,but may be up to 200 mils and thicker. The membrane may be backed by agenerally water impervious layer, such as a Mylar® polyester film(DuPont Teijin Films). When employed, the backing is generally fastenedto the membrane by an adhesive, such as 3M 444 double-sided adhesivetape. Typically, a water impervious backing is used for membranes of lowthickness. A wide variety of polymers may be used provided that they donot bind nonspecifically to the assay components and do not interferewith flow of the sample. Illustrative polymers include polyethylene,polypropylene, polystyrene and the like. Alternatively, the membrane maybe self supporting. Other non-bibulous membranes, such as polyvinylchloride, polyvinyl acetate, copolymers of vinyl acetate and vinylchloride, polyamide, polycarbonate, polystyrene, and the like, can alsobe used. In various embodiments, the label zone material may bepretreated with a solution that includes blocking and stabilizingagents. Blocking agents include bovine serum albumin (BSA), methylatedBSA, casein, nonfat dry milk. The device can also comprise additionalcomponents, including for example buffering agents, HAMA inhibitors,detergents, salts (e.g., chloride and/or sulfate salts of calcium,magnesium, potassium, etc.), and proteinaceous components (e.g., serumalbumin, gelatin, milk proteins, etc.). This list is not meant to belimiting.

The device may further comprise various control locations which are readto determine that the test device has been run properly. By way ofexample, a procedural control zone may be provided separate from theassay detection zone to verify that the sample flow is as expected. Thecontrol zone is preferably a spatially distinct region at which a signalmay be generated that is indicative of the proper flow of reagents. Theprocedural control zone may contain the analyte of interest, or afragment thereof, to which excess labeled antibody used in the analyteassay can bind. In operation, a labeled reagent binds to the controlzone, even when the analyte of interest is absent from the test sample.The use of a control line is helpful in that appearance of a signal inthe control line indicates the time at which the test result can beread, even for a negative result. Thus, when the expected signal appearsin the control line, the presence or absence of a signal in the capturezone can be noted. The device may further comprise a negative controlarea. The purpose of this control area is to alert the user that thetest device is not working properly. When working properly, no signal ormark should be visible in the negative control area.

The outer casing or housing of such an assay device may take variousforms. Typically, it will include an elongate casing and may have aplurality of interfitting parts. In a particularly preferred embodiment,the housing includes a top cover and a bottom support. The top covercontains an application aperture and an observation port. In a preferredembodiment, the housing is made of moisture impervious solid material,for example, a plastic material. It is contemplated that a variety ofcommercially available plastics, including, but not limited to, vinyl,nylon, polyvinyl chloride, polypropylene, polystyrene, polyethylene,polycarbonates, polysulfanes, polyesters, urethanes, and epoxies maybeused to construct a housing. The housing may be prepared by conventionalmethodologies, such as standard molding technologies that are well knownand used in the art. The housing may be produced by molding technologieswhich include, but are not limited to, injection molding, compressionmolding, transfer molding, blow molding, extrusion molding, foammolding, and thermoform molding. The aforementioned molding technologiesare well known in the art and so are not discussed in detail herein. Seefor example, Processes And Materials Of Manufacture, Third Edition, R.A. Lindsberg (1983) Allyn and Baron pp. 393-431.

If necessary, the colorimetric, luminescent, or fluorescent intensity ofthe detectable label being employed may be then evaluated with aninstrument that is appropriate to the label. By way of example, afluorometer can be used to detect fluorescent labels; a reflectometercan be used to detect labels which absorb light, etc. The concentrationof the analyte of interest in the samples may be determined bycorrelating the measured response to the amount of analyte in the samplefluid.

Assay Correlations

The terms “correlating” and “relating” as used herein in reference tothe measurement of biomarkers in an assay refers to determining thepresence, or more preferably the amount, of the biomarker in a samplebased on the signal obtained from the assay. Often, this takes the formof comparing a signal generated from a detectable label on one speciesparticipating in the assay to a predetermined standard curve which canbe used to convert the signal to a concentration or threshold amount ofthe biomarker.

The terms “correlating” and “relating” as used herein in reference tothe use of biomarkers for diagnosis or prognosis refers to comparing thepresence or amount of the biomarker(s) in a patient to its presence oramount in persons known to suffer from, or known to be at risk of, agiven condition; or in persons known to be free of a given condition.Often, this takes the form of comparing an assay result in the form of abiomarker concentration to a predetermined threshold selected to beindicative of the occurrence or nonoccurrence of a disease or thelikelihood of some future outcome.

Selecting a diagnostic threshold involves, among other things,consideration of the probability of disease, distribution of true andfalse diagnoses at different test thresholds, and estimates of theconsequences of treatment (or a failure to treat) based on thediagnosis. For example, when considering administering a specifictherapy which is highly efficacious and has a low level of risk, fewtests are needed because clinicians can accept substantial diagnosticuncertainty. On the other hand, in situations where treatment optionsare less effective and more risky, clinicians often need a higher degreeof diagnostic certainty. Thus, cost/benefit analysis is involved inselecting a diagnostic threshold.

Suitable thresholds may be determined in a variety of ways. For example,one recommended diagnostic threshold for the diagnosis of acutemyocardial infarction using cardiac troponin is the 97.5th percentile ofthe concentration seen in a normal population. Another method may be tolook at serial samples from the same patient, where a prior “baseline”result is used to monitor for temporal changes in a biomarker level.

Population studies may also be used to select a decision threshold.Receiver Operating Characteristic (“ROC”) arose from the field of signaldetection theory developed during World War II for the analysis of radarimages, and ROC analysis is often used to select a threshold able tobest distinguish a “diseased” subpopulation from a “nondiseased”subpopulation. A false positive in this case occurs when the persontests positive, but actually does not have the disease. A falsenegative, on the other hand, occurs when the person tests negative,suggesting they are healthy, when they actually do have the disease. Todraw a ROC curve, the true positive rate (TPR) and false positive rate(FPR) are determined as the decision threshold is varied continuously.Since TPR is equivalent with sensitivity and FPR is equal to1-specificity, the ROC graph is sometimes called the sensitivity vs(1-specificity) plot. A perfect test will have an area under the ROCcurve of 1.0; a random test will have an area of 0.5. A threshold isselected to provide an acceptable level of specificity and sensitivity.

In this context, “diseased” is meant to refer to a population having onecharacteristic (the presence of a disease or condition or the occurrenceof some outcome) and “nondiseased” is meant to refer to a populationlacking the characteristic. While a single decision threshold is thesimplest application of such a method, multiple decision thresholds maybe used. For example, below a first threshold, the absence of diseasemay be assigned with relatively high confidence, and above a secondthreshold the presence of disease may also be assigned with relativelyhigh confidence. Between the two thresholds may be consideredindeterminate. This is meant to be exemplary in nature only.

In addition to threshold comparisons, other methods for correlatingassay results to a patient classification (occurrence or nonoccurrenceof disease, likelihood of an outcome, etc.) include decision trees, rulesets, Bayesian methods, and neural_network methods. These methods canproduce probability values representing the degree to which a subjectbelongs to one classification out of a plurality of classifications.

Measures of test accuracy may be obtained as described in Fischer etal., Intensive Care Med. 29: 1043-51, 2003, and used to determine theeffectiveness of a given biomarker. These measures include sensitivityand specificity, predictive values, likelihood ratios, diagnostic oddsratios, and ROC curve areas. The area under the curve (“AUC”) of a ROCplot is equal to the probability that a classifier will rank a randomlychosen positive instance higher than a randomly chosen negative one. Thearea under the ROC curve may be thought of as equivalent to theMann-Whitney U test, which tests for the median difference betweenscores obtained in the two groups considered if the groups are ofcontinuous data, or to the Wilcoxon test of ranks.

As discussed above, suitable tests may exhibit one or more of thefollowing results on these various measures: a specificity of greaterthan 0.5, preferably at least 0.6, more preferably at least 0.7, stillmore preferably at least 0.8, even more preferably at least 0.9 and mostpreferably at least 0.95, with a corresponding sensitivity greater than0.2, preferably greater than 0.3, more preferably greater than 0.4,still more preferably at least 0.5, even more preferably 0.6, yet morepreferably greater than 0.7, still more preferably greater than 0.8,more preferably greater than 0.9, and most preferably greater than 0.95;a sensitivity of greater than 0.5, preferably at least 0.6, morepreferably at least 0.7, still more preferably at least 0.8, even morepreferably at least 0.9 and most preferably at least 0.95, with acorresponding specificity greater than 0.2, preferably greater than 0.3,more preferably greater than 0.4, still more preferably at least 0.5,even more preferably 0.6, yet more preferably greater than 0.7, stillmore preferably greater than 0.8, more preferably greater than 0.9, andmost preferably greater than 0.95; at least 75% sensitivity, combinedwith at least 75% specificity; a ROC curve area of greater than 0.5,preferably at least 0.6, more preferably 0.7, still more preferably atleast 0.8, even more preferably at least 0.9, and most preferably atleast 0.95; an odds ratio different from 1, preferably at least about 2or more or about 0.5 or less, more preferably at least about 3 or moreor about 0.33 or less, still more preferably at least about 4 or more orabout 0.25 or less, even more preferably at least about 5 or more orabout 0.2 or less, and most preferably at least about 10 or more orabout 0.1 or less; a positive likelihood ratio (calculated assensitivity/(1-specificity)) of greater than 1, at least 2, morepreferably at least 3, still more preferably at least 5, and mostpreferably at least 10; and or a negative likelihood ratio (calculatedas (1-sensitivity)/specificity) of less than 1, less than or equal to0.5, more preferably less than or equal to 0.3, and most preferably lessthan or equal to 0.1

Antibodies

The term “antibody” as used herein refers to a peptide or polypeptidederived from, modeled after or substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof,capable of specifically binding an antigen or epitope. See, e.g.Fundamental Immunology, 3rd Edition, W. E. Paul, ed., Raven Press, N.Y.(1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J.Biochem. Biophys. Methods 25:85-97. The term antibody includesantigen-binding portions, i.e., “antigen binding sites,” (e.g.,fragments, subsequences, complementarity determining regions (CDRs))that retain capacity to bind antigen, including (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR). Singlechain antibodies are also included by reference in the term “antibody.”

Preferred therapeutic antibodies are IgG antibodies. The term “IgG” asused herein is meant a polypeptide belonging to the class of antibodiesthat are substantially encoded by a recognized immunoglobulin gammagene. In humans this class comprises IgG1, IgG2, IgG3, and IgG4. In micethis class comprises IgG1, IgG2a, IgG2b, IgG3. The known Ig domains inthe IgG class of antibodies are VH, Cγ1, Cγ2, Cγ3, VL, and CL. IgG isthe preferred class for therapeutic antibodies for several practicalreasons. IgG antibodies are stable, easily purified, and able to bestored under conditions that are practical for pharmaceutical supplychains. In vivo they have a long biological half-life that is not just afunction of their size but is also a result of their interaction withthe so-called Fc receptor (or FcRn). This receptor seems to protect IgGfrom catabolism within cells and recycles it back to the plasma.

Antibodies are immunological proteins that bind a specific antigen. Inmost mammals, including humans and mice, antibodies are constructed frompaired heavy and light polypeptide chains. The light and heavy chainvariable regions show significant sequence diversity between antibodies,and are responsible for binding the target antigen. Each chain is madeup of individual immunoglobulin (Ig) domains, and thus the generic termimmunoglobulin is used for such proteins.

The term “specifically binds” is not intended to indicate that anantibody binds exclusively to its intended target since, as noted above,an antibody binds to any polypeptide displaying the epitope(s) to whichthe antibody binds. Rather, an antibody “specifically binds” if itsaffinity for its intended target is about 5-fold greater when comparedto its affinity for a non-target molecule which does not display theappropriate epitope(s). Preferably the affinity of the antibody will beat least about 5 fold, preferably 10 fold, more preferably 25-fold, evenmore preferably 50-fold, and most preferably 100-fold or more, greaterfor a target molecule than its affinity for a non-target molecule. Inpreferred embodiments, Preferred antibodies bind with affinities of atleast about 10⁷ M⁻¹, and preferably between about 10⁸ M⁻¹ to about 10⁹M⁻¹, about 10⁹ M⁻¹ to about 10¹⁰ M⁻¹, or about 10¹⁰ M⁻¹ to about 10¹²M⁻¹.

Affinity is calculated as K_(d)=k_(off)/k_(on) (k_(off) is thedissociation rate constant, K_(on) is the association rate constant andK_(d) is the equilibrium constant). Affinity can be determined atequilibrium by measuring the fraction bound (r) of labeled ligand atvarious concentrations (c). The data are graphed using the Scatchardequation: r/c=K(n−r): where r=moles of bound ligand/mole of receptor atequilibrium; c=free ligand concentration at equilibrium; K=equilibriumassociation constant; and n=number of ligand binding sites per receptormolecule. By graphical analysis, r/c is plotted on the Y-axis versus ron the X-axis, thus producing a Scatchard plot. Antibody affinitymeasurement by Scatchard analysis is well known in the art. See, e.g.,van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelson and Griswold,Comput. Methods Programs Biomed. 27: 65-8, 1988.

Antibodies of the invention may be further characterized by epitopemapping, so that antibodies and epitopes may be selected that have thegreatest clinical utility in the immunoassays described herein. The term“epitope” refers to an antigenic determinant capable of specific bindingto an antibody. Epitopes usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents. Preferably, an epitope is targeted which is present on thetarget molecule, but is partially or totally absent on non-targetmolecules.

In some embodiments, the antibody scaffold can be a mixture of sequencesfrom different species. As such, if the antibody is an antibody, suchantibody may be a chimeric antibody and/or a humanized antibody. Ingeneral, both “chimeric antibodies” and “humanized antibodies” refer toantibodies that combine regions from more than one species. For example,“chimeric antibodies” traditionally comprise variable region(s) from amouse (or rat, in some cases) and the constant region(s) from a human.“Humanized antibodies” generally refer to non-human antibodies that havehad the variable-domain framework regions swapped for sequences found inhuman antibodies. Generally, in a humanized antibody, the entireantibody, except the CDRs, is encoded by a polynucleotide of humanorigin or is identical to such an antibody except within its CDRs. TheCDRs, some or all of which are encoded by nucleic acids originating in anon-human organism, are grafted into the beta-sheet framework of a humanantibody variable region to create an antibody, the specificity of whichis determined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525,Verhoeyen et al., 1988, Science 239:1534-1536. “Backmutation” ofselected acceptor framework residues to the corresponding donor residuesis often required to regain affinity that is lost in the initial graftedconstruct (U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762;6,180,370; 5,859,205; 5,821,337; 6,054,297; 6,407,213). The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region, typically that of a humanimmunoglobulin, and thus will typically comprise a human Fc region.Humanized antibodies can also be generated using mice with a geneticallyengineered immune system. Roque et al., 2004, Biotechnol. Prog.20:639-654. A variety of techniques and methods for humanizing andreshaping non-human antibodies are well known in the art (See Tsurushita& Vasquez, 2004, Humanization of Monoclonal Antibodies, MolecularBiology of B Cells, 533-545, Elsevier Science (USA), and referencescited therein). Humanization methods include but are not limited tomethods described in Jones et al., 1986, Nature 321:522-525; Riechmannet al., 1988; Nature 332:323-329; Verhoeyen et al., 1988, Science,239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33;He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, ProcNatl Acad Sci USA 89:4285-9, Presta et al., 1997, Cancer Res.57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci. USA88:4181-4185; O'Connor et al., 1998, Protein Eng 11:321-8. Humanizationor other methods of reducing the immunogenicity of nonhuman antibodyvariable regions may include resurfacing methods, as described forexample in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973.In one embodiment, the parent antibody has been affinity matured, as isknown in the art. Structure-based methods may be employed forhumanization and affinity maturation, for example as described in U.S.Ser. No. 11/004,590. Selection based methods may be employed to humanizeand/or affinity mature antibody variable regions, including but notlimited to methods described in Wu et al., 1999, J. Mol. Biol.294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684;Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al.,1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003,Protein Engineering 16(10):753-759. Other humanization methods mayinvolve the grafting of only parts of the CDRs, including but notlimited to methods described in U.S. Ser. No. 09/810,502; Tan et al.,2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol.169:3076-3084.

In one embodiment, the antibody is a fully human antibody. “Fully humanantibody” or “complete human antibody” refers to a human antibody havingthe gene sequence of an antibody derived from a human chromosome. Fullyhuman antibodies may be obtained, for example, using transgenic mice(Bruggemann et al., 1997, Curr Opin Biotechnol 8:455-458) or humanantibody libraries coupled with selection methods (Griffiths et al.,1998, Curr Opin Biotechnol 9:102-108).

Production of Antibodies

Monoclonal antibody preparations can be produced using a wide variety oftechniques known in the art including the use of hybridoma, recombinant,and phage display technologies, or a combination thereof. For example,monoclonal antibodies can be produced using hybridoma techniquesincluding those known in the art and taught, for example, in Harlow etal., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: MONOCLONAL ANTIBODIES ANDT-CELL HYBRIDOMAS, pp. 563-681 (Elsevier, N.Y., 1981) (both of which areincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

Monoclonal antibodies derived from animals other than rats and miceoffer unique advantages. Many protein targets relevant to signaltransduction and disease are highly conserved between mice, rats andhumans, and can therefore be recognized as self-antigens by a mouse orrat host, making them less immunogenic. This problem may be avoided whenusing rabbit as a host animal. See, e.g., Rossi et al., Am. J. Clin.Pathol., 124, 295-302, 2005.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anon-limiting example, mice can be immunized with an antigen of interestor a cell expressing such an antigen. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells. Hybridomas are selected and cloned by limiting dilution.The hybridoma clones are then assayed by methods known in the art forcells that secrete antibodies capable of binding the antigen. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by inoculating mice intraperitoneally with positive hybridomaclones.

Adjuvants that can be used in the methods of antibody generationinclude, but are not limited to, protein adjuvants; bacterial adjuvants,e.g., whole bacteria (BCG, Corynebacterium parvum, Salmonella minnesota)and bacterial components including cell wall skeleton, trehalosedimycolate, monophosphoryl lipid A, methanol extractable residue (MER)of tubercle bacillus, complete or incomplete Freund's adjuvant; viraladjuvants; chemical adjuvants, e.g., aluminum hydroxide, iodoacetate andcholesteryl hemisuccinateor; naked DNA adjuvants. Other adjuvants thatcan be used in the methods of the invention include, Cholera toxin,paropox proteins, MF-59 (Chiron Corporation; See also Bieg et al. (1999)“GAD65 And Insulin B Chain Peptide (9-23) Are Not Primary AutoantigensIn The Type 1 Diabetes Syndrome Of The BB Rat,” Autoimmunity,31(1):15-24, which is incorporated herein by reference), MPL® (CorixaCorporation; See also Lodmell et al. (2000) “DNA Vaccination Of MiceAgainst Rabies Virus: Effects Of The Route Of Vaccination And TheAdjuvant Monophosphoryl Lipid A (MPL),” Vaccine, 18: 1059-1066; Johnsonet al. (1999) “3-O-Desacyl Monophosphoryl Lipid A Derivatives: SynthesisAnd Immunostimulant Activities,” Journal of Medicinal Chemistry, 42:4640-4649; Baldridge et al. (1999) “Monophosphoryl Lipid A (MPL)Formulations For The Next Generation Of Vaccines,” Methods, 19: 103-107,all of which are incorporated herein by reference), RC-529 adjuvant(Corixa Corporation; the lead compound from Corixa's aminoalkylglucosaminide 4-phosphate (AGP) chemical library, see alsowww.corixa.com), and DETOX™ adjuvant (Corixa Corporation; DETOX™adjuvant includes MPL® adjuvant (monophosphoryl lipid A) andmycobacterial cell wall skeleton; See also Eton et al. (1998) “ActiveImmunotherapy With Ultraviolet B-Irradiated Autologous Whole MelanomaCells Plus DETOX In Patients With Metastatic Melanoma,” Clin. CancerRes. 4(3):619-627; and Gupta et al. (1995) “Adjuvants For HumanVaccines—Current Status, Problems And Future Prospects,” Vaccine,13(14): 1263-1276, both of which are incorporated herein by reference).

Numerous publications discuss the use of phage display technology toproduce and screen libraries of polypeptides for binding to a selectedanalyte. See, e.g, Cwirla et al., Proc. Natl. Acad. Sci. USA 87,6378-82, 1990; Devlin et al., Science 249, 404-6, 1990, Scott and Smith,Science 249, 386-88, 1990; and Ladner et al., U.S. Pat. No. 5,571,698. Abasic concept of phage display methods is the establishment of aphysical association between DNA encoding a polypeptide to be screenedand the polypeptide. This physical association is provided by the phageparticle, which displays a polypeptide as part of a capsid enclosing thephage genome which encodes the polypeptide. The establishment of aphysical association between polypeptides and their genetic materialallows simultaneous mass screening of very large numbers of phagebearing different polypeptides. Phage displaying a polypeptide withaffinity to a target bind to the target and these phage are enriched byaffinity screening to the target. The identity of polypeptides displayedfrom these phage can be determined from their respective genomes. Usingthese methods a polypeptide identified as having a binding affinity fora desired target can then be synthesized in bulk by conventional means.See, e.g., U.S. Pat. No. 6,057,098, which is hereby incorporated in itsentirety, including all tables, figures, and claims.

The antibodies that are generated by these methods may then be selectedby first screening for affinity and specificity with the purifiedpolypeptide of interest and, if required, comparing the results to theaffinity and specificity of the antibodies with polypeptides that aredesired to be excluded from binding. The screening procedure can involveimmobilization of the purified polypeptides in separate wells ofmicrotiter plates. The solution containing a potential antibody orgroups of antibodies is then placed into the respective microtiter wellsand incubated for about 30 min to 2 h. The microtiter wells are thenwashed and a labeled secondary antibody (for example, an anti-mouseantibody conjugated to alkaline phosphatase if the raised antibodies aremouse antibodies) is added to the wells and incubated for about 30 minand then washed. Substrate is added to the wells and a color reactionwill appear where antibody to the immobilized polypeptide(s) arepresent.

The antibodies so identified may then be further analyzed for affinityand specificity in the assay design selected. In the development ofimmunoassays for a target protein, the purified target protein acts as astandard with which to judge the sensitivity and specificity of theimmunoassay using the antibodies that have been selected. Because thebinding affinity of various antibodies may differ; certain antibodypairs (e.g., in sandwich assays) may interfere with one anothersterically, etc., assay performance of an antibody may be a moreimportant measure than absolute affinity and specificity of an antibody.

Antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized using conventional methodologies with a selected antigen,e.g., all or a portion of a polypeptide of the invention. Monoclonalantibodies directed against the antigen can be obtained from theimmunized, transgenic mice using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies. For an overview of this technology for producing humanantibodies, see Lonberg et al. (1995) “Human Antibodies From TransgenicMice,” Int. Rev. Immunol. 13:65-93, which is incorporated herein byreference in its entirety). For a detailed discussion of this technologyfor producing human antibodies and human monoclonal antibodies andprotocols for producing such antibodies, see, e.g., InternationalPublication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S.Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016,5,545,806, 5,814,318, and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.) and Medarex (Princeton, N.J.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

Recombinant Expression of Antibodies

Once a nucleic acid sequence encoding an antibody of the invention hasbeen obtained, the vector for the production of the antibody may beproduced by recombinant DNA technology using techniques well known inthe art. Methods which are well known to those skilled in the art can beused to construct expression vectors containing the antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.(See, for example, the techniques described in Sambrook et al, 1990,MOLECULAR CLONING, A LABORATORY MANUAL, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. and Ausubel et al. eds., 1998,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y.).

An expression vector comprising the nucleotide sequence of an antibodycan be transferred to a host cell by conventional techniques (e.g.,electroporation, liposomal transfection, and calcium phosphateprecipitation) and the transfected cells are then cultured byconventional techniques to produce the antibody of the invention. Inspecific embodiments, the expression of the antibody is regulated by aconstitutive, an inducible or a tissue, specific promoter.

The anti-TIMP2 antibodies disclosed herein may also be producedrecombinantly (e.g., in an E. coli/T7 expression system, a mammaliancell expression system or a lower eukaryote expression system). In thisembodiment, nucleic acids encoding the antibody immunoglobulin moleculesof the invention (e.g., V_(H) or V_(L)) may be inserted into a pET-basedplasmid and expressed in the E. coli/T7 system. For example, the presentinvention includes methods for expressing an antibody or antigen-bindingfragment thereof or immunoglobulin chain thereof in a host cell (e.g.,bacterial host cell such as E. coli such as BL21 or BL21DE3) comprisingexpressing T7 RNA polymerase in the cell which also includes apolynucleotide encoding an immunoglobulin chain that is operably linkedto a T7 promoter. For example, in an embodiment of the invention, abacterial host cell, such as a E. coli, includes a polynucleotideencoding the T7 RNA polymerase gene operably linked to a lac promoterand expression of the polymerase and the chain is induced by incubationof the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside).

Thus, the present invention includes recombinant methods for making ananti-TIMP2 antibody or antigen-binding fragment thereof of the presentinvention, or an immunoglobulin chain thereof, comprising introducing apolynucleotide encoding one or more immunoglobulin chains of theantibody or fragment (e.g., heavy and/or light immunoglobulin chain);culturing the host cell (e.g., CHO or Pichia or Pichia pastoris) undercondition favorable to such expression and, optionally, isolating theantibody or fragment or chain from the host cell and/or medium in whichthe host cell is grown.

Anti-TIMP2 antibodies can also be synthesized by any of the methods setforth in U.S. Pat. No. 6,331,415.

Eukaryotic and prokaryotic host cells, including mammalian cells ashosts for expression of the antibodies or fragments or immunoglobulinchains disclosed herein are well known in the art and include manyimmortalized cell lines available from the American Type CultureCollection (ATCC). These include, inter alia, Chinese hamster ovary(CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK)cells, monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number ofother cell lines. Mammalian host cells include human, mouse, rat, dog,monkey, pig, goat, bovine, horse and hamster cells. Cell lines ofparticular preference are selected through determining which cell lineshave high expression levels. Other cell lines that may be used areinsect cell lines, such as Sf9 cells, amphibian cells, bacterial cells,plant cells and fungal cells. Fungal cells include yeast and filamentousfungus cells including, for example, Pichia pastoris, Pichia finlandica,Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichiaminuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp.,Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporiumlucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum,Physcomitrella patens and Neurospora crassa. Pichia sp., anySaccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candidaalbicans, any Aspergillus sp., Trichoderma reesei, Chrysosporiumlucknowense, any Fusarium sp., Yarrowia lipolytica, and Neurosporacrassa. When recombinant expression vectors encoding the heavy chain orantigen-binding portion or fragment thereof, the light chain and/orantigen-binding fragment thereof are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody orfragment or chain in the host cells or secretion of the into the culturemedium in which the host cells are grown.

A variety of host-expression vector systems may be utilized to expressthe antibodies of the invention. Such host-expression systems representvehicles by which the coding sequences of the antibodies may be producedand subsequently purified, but also represent cells which may, whentransformed or transfected with the appropriate nucleotide codingsequences, express the antibodies of the invention in situ. Theseinclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing immunoglobulincoding sequences; yeast (e.g., Saccharomyces pichia) transformed withrecombinant yeast expression vectors containing immunoglobulin codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the immunoglobulincoding sequences; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus (CaMV) and tobaccomosaic virus (TMV)) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasmid) containing immunoglobulin coding sequences;or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells,lymphotic cells (see U.S. Pat. No. 5,807,715), Per C.6 cells (ratretinal cells developed by Crucell)) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodybeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of pharmaceutical compositions of anantibody, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited, to the E. coli expression vectorpUR278 (Ruther et al. (1983) “Easy Identification Of cDNA Clones,” EMBOJ. 2:1791-1794), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye et al. (1985)“Up-Promoter Mutations In The Lpp Gene Of Escherichia coli,” NucleicAcids Res. 13:3101-3110; Van Heeke et al. (1989) “Expression Of HumanAsparagine Synthetase In Escherichia coli,” J. Biol. Chem.24:5503-5509); and the like. pGEX vectors may also be used to expressforeign polypeptides as fusion proteins with glutathione S-transferase(GST). In general, such fusion proteins are soluble and can easily bepurified from lysed cells by adsorption and binding to a matrixglutathione-agarose beads followed by elution in the presence of freegluta-thione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (e.g., the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (e.g., thepolyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the immunoglobulin molecule in infected hosts. (see e.g., seeLogan et al. (1984) “Adenovirus Tripartite Leader Sequence EnhancesTranslation Of mRNAs Late After Infection,” Proc. Natl. Acad. Sci.(U.S.A.) 81:3655-3659). Specific initiation signals may also be requiredfor efficient translation of inserted antibody coding sequences. Thesesignals include the ATG initiation codon and adjacent sequences.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (seeBitter et al. (1987) “Expression And Secretion Vectors For Yeast,”Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 andHs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably express anantibody of the invention may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express theantibodies of the invention. Such engineered cell lines may beparticularly useful in screening and evaluation of compounds thatinteract directly or indirectly with the antibodies of the invention.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al. (1977)“Transfer Of Purified Herpes Virus Thymidine Kinase Gene To CulturedMouse Cells,” Cell 11:223-232), hypoxanthine-guaninephosphoribosyltransferase (Szybalska et al. (1962) “Genetics Of HumanCess Line. IV. DNA-Mediated Heritable Transformation Of A BiochemicalTrait,” Proc. Natl. Acad. Sci. (U.S.A.) 48:2026-2034), and adeninephosphoribosyltransferase (Lowy et al. (1980) “Isolation Of TransformingDNA: Cloning The Hamster Aprt Gene,” Cell 22:817-823) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al. (1980) “Transformation Of Mammalian Cells With An AmplifiableDominant-Acting Gene,” Proc. Natl. Acad. Sci. (U.S.A.) 77:3567-3570;O'Hare et al. (1981) “Transformation Of Mouse Fibroblasts ToMethotrexate Resistance By A Recombinant Plasmid Expressing AProkaryotic Dihydrofolate Reductase,” Proc. Natl. Acad. Sci. (U.S.A.)78:1527-1531); gpt, which confers resistance to mycophenolic acid(Mulligan et al. (1981) “Selection For Animal Cells That Express TheEscherichia coli Gene Coding For Xanthine-GuaninePhosphoribosyltransferase,” Proc. Natl. Acad. Sci. (U.S.A.)78:2072-2076); neo, which confers resistance to the aminoglycoside G-418(Tachibana et al. (1991) “Altered Reactivity Of Immunoglobutin ProducedBy Human-Human Hybridoma Cells Transfected By pSV2-Neo Gene,”Cytotechnology 6(3):219-226; Tolstoshev (1993) “Gene Therapy, Concepts,Current Trials And Future Directions,” Ann. Rev. Pharmacol. Toxicol.32:573-596; Mulligan (1993) “The Basic Science Of Gene Therapy,” Science260:926-932; and Morgan et al. (1993) “Human gene therapy,” Ann. Rev.Biochem. 62:191-217). Methods commonly known in the art of recombinantDNA technology which can be used are described in Ausubel et al. (eds.),1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y.;Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL,Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds),1994, CURRENT PROTOCOLS IN HUMAN GENETICS, John Wiley & Sons, N.Y.;Colbere-Garapin et al. (1981) “A New Dominant Hybrid Selective MarkerFor Higher Eukaryotic Cells,” J. Mol. Biol. 150:1-14; and hygro, whichconfers resistance to hygromycin (Santerre et al. (1984) “Expression OfProkaryotic Genes For Hygromycin B And G418 Resistance AsDominant-Selection Markers In Mouse L Cells,” Gene 30:147-156).

The expression levels of an antibody of the invention can be increasedby vector amplification (for a review, see Bebbington and Hentschel,“The Use Of Vectors Based On Gene Amplification For The Expression OfCloned Genes In Mammaian Cells,” in DNA CLONING, Vol. 3. (AcademicPress, N.Y., 1987)). When a marker in the vector system expressing anantibody is amplifiable, increase in the level of inhibitor present inculture of host cell will increase the number of copies of the markergene. Since the amplified region is associated with the nucleotidesequence of the antibody, production of the antibody will also increase(Crouse et al. (1983) “Expression And Amplification Of Engineered MouseDihydrofolate Reductase Minigenes,” Mol. Cell. Biol. 3:257-266).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot (1986) “Expression And Amplification Of Engineered MouseDihydrofolate Reductase Minigenes,” Nature 322:562-565; Kohler (1980)“Immunoglobulin Chain Loss In Hybridoma Lines,” Proc. Natl. Acad. Sci.(U.S.A.) 77:2197-2199). The coding sequences for the heavy and lightchains may comprise cDNA or genomic DNA.

In general, glycoproteins produced in a particular cell line ortransgenic animal will have a glycosylation pattern that ischaracteristic for glycoproteins produced in the cell line or transgenicanimal. Therefore, the particular glycosylation pattern of an antibodywill depend on the particular cell line or transgenic animal used toproduce the antibody. However, all antibodies encoded by the nucleicacid molecules provided herein, or comprising the amino acid sequencesprovided herein, comprise the instant invention, independent of theglycosylation pattern the antibodies may have. Similarly, in particularembodiments, antibodies with a glycosylation pattern comprising onlynon-fucosylated N-glycans may be advantageous, because these antibodieshave been shown to typically exhibit more potent efficacy than theirfucosylated counterparts both in vitro and in vivo (See for example,Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Pat. Nos.6,946,292 and 7,214,775). These antibodies with non-fucosylatedN-glycans are not likely to be immunogenic because their carbohydratestructures are a normal component of the population that exists in humanserum IgG.

Once the antibody of the invention has been recombinantly expressed, itmay be purified by any method known in the art for purification of anantibody, for example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen after Protein A, andsizing column chromatography), centrifugation, differential solubility,or by any other standard technique for the purification of proteins.

Antibody Conjugates

The anti-TIMP2 antibodies and antigen-binding fragments thereofdisclosed herein may also be conjugated to a chemical moiety. Thechemical moiety may be, inter alia, a polymer, a radionuclide or acytotoxic factor. In particular embodiments, the chemical moiety is apolymer which increases the half-life of the antibody or fragment in thebody of a subject. Suitable polymers include, but are not limited to,hydrophilic polymers which include but are not limited to polyethyleneglycol (PEG) (e.g., PEG with a molecular weight of 2 kDa, 5 kDa, 10 kDa,12 kDa, 20 kDa, 30 kDa or 40 kDa), dextran and monomethoxypolyethyleneglycol (mPEG). Lee, et al., (1999) (Bioconj. Chem. 10:973-981) disclosesPEG conjugated single-chain antibodies. Wen, et al., (2001) (Bioconj.Chem. 12:545-553) disclose conjugating antibodies with PEG which isattached to a radiometal chelator (diethylenetriaminpentaacetic acid(DTPA)).

The antibodies and antigen-binding fragments thereof disclosed hereinmay also be conjugated with labels such as ⁹⁹Tc, ⁹⁰Y, ¹¹¹In, ³²P, ¹⁴C,¹²⁵I, ³H, ¹³¹I, ¹¹C, ¹⁵O, ¹³N, ¹⁸F, ³⁵S, ⁵¹Cr, ⁵⁷To, ²²⁶Ra, ⁶⁰Co, ⁵⁹Fe,⁵⁷Se, ¹⁵²Eu, ⁶⁷CU, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, ²³⁴Th, and ⁴⁰K,¹⁵⁷Gd, ⁵⁵Mn, ⁵²Tr, and ⁵⁶Fe.

The antibodies and antigen-binding fragments disclosed herein may alsobe PEGylated, for example to increase its biological (e.g., serum)half-life. To PEGylate an antibody or fragment, the antibody orfragment, typically is reacted with a reactive form of polyethyleneglycol (PEG), such as a reactive ester or aldehyde derivative of PEG,under conditions in which one or more PEG groups become attached to theantibody or antibody fragment. In particular embodiments, the PEGylationis carried out via an acylation reaction or an alkylation reaction witha reactive PEG molecule (or an analogous reactive water-solublepolymer). As used herein, the term “polyethylene glycol” is intended toencompass any of the forms of PEG that have been used to derivatizeother proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethyleneglycol or polyethylene glycol-maleimide. In certain embodiments, theantibody or fragment to be PEGylated is an aglycosylated antibody orfragment. Methods for PEGylating proteins are known in the art and canbe applied to the antibodies of the invention. See, e.g., EP 0 154 316and EP 0 401 384.

The antibodies and antigen-binding fragments disclosed herein may alsobe conjugated with fluorescent or chemilluminescent labels, includingfluorophores such as rare earth chelates, fluorescein and itsderivatives, rhodamine and its derivatives, isothiocyanate,phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde,fluorescamine, ¹⁵²Eu, dansyl, umbelliferone, luciferin, luminal label,isoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridimium salt label, an oxalate ester label, an aequorinlabel, 2,3-dihydrophthalazinediones, biotin/avidin, spin labels andstable free radicals.

The antibodies and antigen-binding fragments thereof of the inventionmay also be conjugated to a cytotoxic factor such as diptheria toxin,Pseudomonas aeruginosa exotoxin A chain, ricin A chain, abrin A chain,modeccin A chain, alpha-sarcin, Aleurites fordii proteins and compounds(e.g., fatty acids), dianthin proteins, Phytoiacca americana proteinsPAPI, PAPII, and PAP-S, Momordica charantia inhibitor, curcin, crotin,Saponaria officinalis inhibitor, mitogellin, restrictocin, phenomycin,and enomycin.

Any method known in the art for conjugating the antibodies andantigen-binding fragments thereof of the invention to the variousmoieties may be employed, including those methods described by Hunter,et al., (1962) Nature 144:945; David, et al., (1974) Biochemistry13:1014; Pain, et al., (1981) J. Immunol. Meth. 40:219; and Nygren, J.,(1982) Histochem. and Cytochem. 30:407. Methods for conjugatingantibodies and fragments are conventional and very well known in theart.

Therapeutic Uses of Anti-TIMP2 Antibodies

Further provided are methods for treating subjects, including humansubjects, in need of treatment with the isolated antibodies orantigen-binding fragments thereof disclosed herein.

To prepare pharmaceutical or sterile compositions of the anti-TIMP2antibodies and antigen-binding fragments of the invention (e.g.,antibody 6E2.1 or 40H2-40K3 and humanized versions thereof), theantibody or antigen-binding fragment thereof is admixed with apharmaceutically acceptable carrier or excipient. See, e.g., Remington'sPharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, MackPublishing Company, Easton, Pa. (1984).

Formulations of therapeutic and diagnostic agents may be prepared bymixing with acceptable carriers, excipients, or stabilizers in the formof, e.g., lyophilized powders, slurries, aqueous solutions orsuspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's ThePharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;Gennaro (2000) Remington: The Science and Practice of Pharmacy,Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.)(1993) Pharmaceutical Dosage Forms: Parenteral Medications, MarcelDekker, N.Y.; Lieberman, et al. (eds.) (1990) Pharmaceutical DosageForms: Tablets, Marcel Dekker, N.Y.; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, N.Y.;Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, MarcelDekker, Inc., New York, N.Y.).

Toxicity and therapeutic efficacy of the antibodies of the invention,administered alone or in combination with another therapeutic agent, canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index (LD₅₀/ED₅₀). The data obtained fromthese cell culture assays and animal studies can be used in formulatinga range of dosage for use in human. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administration.

In a further embodiment, a further therapeutic agent that isadministered to a subject in association with an anti-TIMP2 antibody orantigen-binding fragment thereof of the invention (e.g., antibody 131Aor humanized versions thereof) in accordance with the Physicians' DeskReference 2003 (Thomson Healthcare; 57th edition (Nov. 1, 2002)).

The mode of administration can vary. Routes of administration includeoral, rectal, transmucosal, intestinal, parenteral; intramuscular,subcutaneous, intradermal, intramedullary, intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, intraocular,inhalation, insufflation, topical, cutaneous, transdermal, orintra-arterial.

In particular embodiments, the anti-TIMP2 antibodies or antigen-bindingfragments thereof of the invention (e.g., antibody 131A and humanizedversions thereof) can be administered by an invasive route such as byinjection. In further embodiments of the invention, an anti-TIMP2antibody or antigen-binding fragment thereof, or pharmaceuticalcomposition thereof, is administered intravenously, subcutaneously,intramuscularly, intraarterially, intratumorally, or by inhalation,aerosol delivery. Administration by non-invasive routes (e.g., orally;for example, in a pill, capsule or tablet) is also within the scope ofthe present invention.

The present invention provides a vessel (e.g., a plastic or glass vial,e.g., with a cap or a chromatography column, hollow bore needle or asyringe cylinder) comprising any of the antibodies or antigen-bindingfragments of the invention (e.g., antibody 131A and humanized versionsthereof) or a pharmaceutical composition thereof. The present inventionalso provides an injection device comprising any of the antibodies orantigen-binding fragments of the invention (e.g., antibody 131A andhumanized versions thereof) or a pharmaceutical composition thereof. Aninjection device is a device that introduces a substance into the bodyof a patient via a parenteral route, e.g., intramuscular, subcutaneousor intravenous. For example, an injection device may be a syringe (e.g.,pre-filled with the pharmaceutical composition, such as anauto-injector) which, for example, includes a cylinder or barrel forholding fluid to be injected (e.g., antibody or fragment or apharmaceutical composition thereof), a needle for piecing skin and/orblood vessels for injection of the fluid; and a plunger for pushing thefluid out of the cylinder and through the needle bore. In an embodimentof the invention, an injection device that comprises an antibody orantigen-binding fragment thereof of the present invention or apharmaceutical composition thereof is an intravenous (IV) injectiondevice. Such a device includes the antibody or fragment or apharmaceutical composition thereof in a cannula or trocar/needle whichmay be attached to a tube which may be attached to a bag or reservoirfor holding fluid (e.g., saline; or lactated ringer solution comprisingNaCl, sodium lactate, KCl, CaCl₂ and optionally including glucose)introduced into the body of the patient through the cannula ortrocar/needle. The antibody or fragment or a pharmaceutical compositionthereof may, in an embodiment of the invention, be introduced into thedevice once the trocar and cannula are inserted into the vein of asubject and the trocar is removed from the inserted cannula. The IVdevice may, for example, be inserted into a peripheral vein (e.g., inthe hand or arm); the superior vena cava or inferior vena cava, orwithin the right atrium of the heart (e.g., a central IV); or into asubclavian, internal jugular, or a femoral vein and, for example,advanced toward the heart until it reaches the superior vena cava orright atrium (e.g., a central venous line). In an embodiment of theinvention, an injection device is an autoinjector; a jet injector or anexternal infusion pump. A jet injector uses a high-pressure narrow jetof liquid which penetrate the epidermis to introduce the antibody orfragment or a pharmaceutical composition thereof to a patient's body.External infusion pumps are medical devices that deliver the antibody orfragment or a pharmaceutical composition thereof into a patient's bodyin controlled amounts. External infusion pumps may be poweredelectrically or mechanically. Different pumps operate in different ways,for example, a syringe pump holds fluid in the reservoir of a syringe,and a moveable piston controls fluid delivery, an elastomeric pump holdsfluid in a stretchable balloon reservoir, and pressure from the elasticwalls of the balloon drives fluid delivery. In a peristaltic pump, a setof rollers pinches down on a length of flexible tubing, pushing fluidforward. In a multi-channel pump, fluids can be delivered from multiplereservoirs at multiple rates.

The pharmaceutical compositions disclosed herein may also beadministered with a needleless hypodermic injection device; such as thedevices disclosed in U.S. Pat. Nos. 6,620,135; 6,096,002; 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556. Suchneedleless devices comprising the pharmaceutical composition are alsopart of the present invention. The pharmaceutical compositions disclosedherein may also be administered by infusion. Examples of well-knownimplants and modules for administering the pharmaceutical compositionsinclude those disclosed in: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,447,233, which discloses a medicationinfusion pump for delivering medication at a precise infusion rate; U.S.Pat. No. 4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments. Many other such implants, delivery systems, and modulesare well known to those skilled in the art and those comprising thepharmaceutical compositions of the present invention are within thescope of the present invention.

Alternately, one may administer the anti-TIMP2 antibody orantigen-binding fragment of the invention (e.g., antibody 131A andhumanized versions thereof) in a local rather than systemic manner, forexample, via injection of the antibody or fragment directly into atumor, e.g., a TIMP2⁺ tumor. Furthermore, one may administer theantibody or fragment in a targeted drug delivery system, for example, ina liposome coated with a tissue-specific antibody, targeting, forexample, a tumor e.g., a TIMP2⁺ tumor, e.g., characterized byimmunopathology. The liposomes will be targeted to and taken upselectively by the afflicted tissue. Such methods and liposomes are partof the present invention.

The administration regimen depends on several factors, including theserum or tissue turnover rate of the therapeutic antibody orantigen-binding fragment, the level of symptoms, the immunogenicity ofthe therapeutic antibody, and the accessibility of the target cells inthe biological matrix. Preferably, the administration regimen deliverssufficient therapeutic antibody or fragment to effect improvement in thetarget disease state, while simultaneously minimizing undesired sideeffects. Accordingly, the amount of biologic delivered depends in parton the particular therapeutic antibody and the severity of the conditionbeing treated. Guidance in selecting appropriate doses of therapeuticantibodies or fragments is available (see, e.g., Wawrzynczak (1996)Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina(ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, MarcelDekker, New York, N.Y.; Bach (ed.) (1993) Monoclonal Antibodies andPeptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.;Baert, et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al.(1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl.J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med.342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky etal. (2000) New Engl. J. Med. 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment. Generally, the dose begins with an amount somewhat less thanthe optimum dose and it is increased by small increments thereafteruntil the desired or optimum effect is achieved relative to any negativeside effects. Important diagnostic measures include those of symptomsof, e.g., the inflammation or level of inflammatory cytokines produced.In general, it is desirable that a biologic that will be used is derivedfrom the same species as the animal targeted for treatment, therebyminimizing any immune response to the reagent. In the case of humansubjects, for example, humanized and fully human antibodies may bedesirable.

Antibodies or antigen-binding fragments thereof disclosed herein (e.g.,antibody 131A and humanized versions thereof) may be provided bycontinuous infusion, or by doses administered, e.g., daily, 1-7 timesper week, weekly, bi-weekly, monthly, bimonthly, quarterly,semiannually, annually etc. Doses may be provided, e.g., intravenously,subcutaneously, topically, orally, nasally, rectally, intramuscular,intracerebrally, intraspinally, or by inhalation. A total weekly dose isgenerally at least 0.05 μg/kg body weight, more generally at least 0.2μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.25 mg/kg, 1.0 mg/kg,2.0 mg/kg, 5.0 mg/ml, 10 mg/kg, 25 mg/kg, 50 mg/kg or more (see, e.g.,Yang, et al. (2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002)New Engl. J. Med. 346:1692-1698; Liu, et al. (1999) J. Neurol.Neurosurg. Psych. 67:451-456; Portielji, et al. (20003) Cancer Immunol.Immunother. 52:151-144). Doses may also be provided to achieve apre-determined target concentration of anti-TIMP2 antibody in thesubject's serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg/ml or more.In other embodiments, An anti-TIMP2 antibody of the present invention isadministered, e.g., subcutaneously or intravenously, on a weekly,biweekly, “every 4 weeks,” monthly, bimonthly, or quarterly basis at 10,20, 50, 80, 100, 200, 500, 1000 or 2500 mg/subject.

As used herein, the term “effective amount” refer to an amount of ananti-TIMP2 or antigen-binding fragment thereof of the invention (e.g.,antibody 131A and humanized versions thereof) that, when administeredalone or in combination with an additional therapeutic agent to a cell,tissue, or subject, is effective to cause a measurable improvement inone or more symptoms of disease, for example cancer or the progressionof cancer. An effective dose further refers to that amount of theantibody or fragment sufficient to result in at least partialamelioration of symptoms, e.g., tumor shrinkage or elimination, lack oftumor growth, increased survival time. When applied to an individualactive ingredient administered alone, an effective dose refers to thatingredient alone. When applied to a combination, an effective doserefers to combined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously. An effective amount of a therapeutic will result in animprovement of a diagnostic measure or parameter by at least 10%;usually by at least 20%; preferably at least about 30%; more preferablyat least 40%, and most preferably by at least 50%. An effective amountcan also result in an improvement in a subjective measure in cases wheresubjective measures are used to assess disease severity.

Experimental and Diagnostic Uses

The anti-TIMP2 antibodies and antigen-binding fragments thereofdisclosed herein may be used as affinity purification agents. In thisprocess, the anti-TIMP2 antibodies and antigen-binding fragments thereofare immobilized on a solid phase such a Sephadex, glass or agarose resinor filter paper, using methods well known in the art. The immobilizedantibody or fragment is contacted with a sample containing the TIMP2protein (or a fragment thereof) to be purified, and thereafter thesupport is washed with a suitable solvent that will remove substantiallyall the material in the sample except the TIMP2 protein, which is boundto the immobilized antibody or fragment. Finally, the support is washedwith a solvent which elutes the bound TIMP2 (e.g., protein A). Suchimmobilized antibodies and fragments form part of the present invention.

Further provided are antigens for generating secondary antibodies whichare useful for example for performing Western blots and otherimmunoassays discussed herein.

Anti-TIMP2 antibodies (e.g., humanized antibodies) and antigen-bindingfragments thereof may also be useful in diagnostic assays for TIMP2protein, e.g., detecting its expression in specific cells, tissues, orserum, e.g., tumor cells such as melanoma cells. Such diagnostic methodsmay be useful in various disease diagnoses.

The present invention includes ELISA assays (enzyme-linked immunosorbentassay) incorporating the use of an anti-TIMP2 antibody orantigen-binding fragment thereof disclosed herein (e.g., antibody 131Aor a humanized version thereof).

For example, such a method comprises the following steps:

-   -   (a) coat a substrate (e.g., surface of a microtiter plate well,        e.g., a plastic plate) with anti-TIMP2 antibody or        antigen-binding fragment thereof;    -   (b) apply a sample to be tested for the presence of TIMP2 to the        substrate;    -   (c) wash the plate, so that unbound material in the sample is        removed;    -   (d) apply detectably labeled antibodies (e.g., enzyme-linked        antibodies) which are also specific to the TIMP2 antigen;    -   (e) wash the substrate, so that the unbound, labeled antibodies        are removed;    -   (f) if the labeled antibodies are enzyme linked, apply a        chemical which is converted by the enzyme into a fluorescent        signal; and    -   (g) detect the presence of the labeled antibody.        Detection of the label associated with the substrate indicates        the presence of the TIMP2 protein.

In a further embodiment, the labeled antibody or antigen-bindingfragment thereof is labeled with peroxidase which react with ABTS (e.g.,2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) or3,3′,5,5′-Tetramethylbenzidine to produce a color change which isdetectable. Alternatively, the labeled antibody or fragment is labeledwith a detectable radioisotope (e.g., ³H) which can be detected byscintillation counter in the presence of a scintillant.

An anti-TIMP2 antibody or antigen-binding fragment thereof of theinvention may be used in a Western blot or immune-protein blotprocedure. Such a procedure forms part of the present invention andincludes e.g., optionally transferring proteins from a sample to betested for the presence of TIMP2 (e.g., from a PAGE or SDS-PAGEelectrophoretic separation of the proteins in the sample) onto amembrane or other solid substrate using a method known in the art (e.g.,semi-dry blotting or tank blotting); contacting the membrane or othersolid substrate to be tested for the presence of bound TIMP2 or afragment thereof with an anti-TIMP2 antibody or antigen-binding fragmentthereof of the invention; washing the membrane one or more times toremove unbound anti-TIMP2 antibody or fragment and other unboundsubstances; and detecting the bound anti-TIMP2 antibody or fragment.

Such a membrane may take the form of a nitrocellulose or vinyl-based(e.g., polyvinylidene fluoride (PVDF)) membrane to which the proteins tobe tested for the presence of TIMP2 in a non-denaturing PAGE(polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) gel have been transferred(e.g., following electrophoretic separation in the gel). Beforecontacting the membrane with the anti-TIMP2 antibody or fragment, themembrane is optionally blocked, e.g., with non-fat dry milk or the likeso as to bind non-specific protein binding sites on the membrane.

Detection of the bound antibody or fragment indicates that the TIMP2protein is present on the membrane or substrate and in the sample.Detection of the bound antibody or fragment may be by binding theantibody or fragment with a secondary antibody (an anti-immunoglobulinantibody) which is detectably labeled and, then, detecting the presenceof the secondary antibody.

The anti-TIMP2 antibodies and antigen-binding fragments thereofdisclosed herein may also be used for immunohistochemistry. Such amethod forms part of the present invention and comprises, e.g.,contacting a cell (e.g., a tumor cell such as a melanoma cell) to betested for the presence of TIMP2 protein with an anti-TIMP2 antibody orantigen-binding fragment thereof of the invention; and detecting theantibody or fragment on or in the cell.

If the antibody or fragment itself is detectably labeled, it can bedetected directly. Alternatively, the antibody or fragment may be boundby a detectably labeled secondary antibody which is detected.

Certain anti-TIMP2 antibodies and antigen-binding fragments thereofdisclosed herein may also be used for in vivo tumor imaging. Such amethod may include injection of a radiolabeled anti-TIMP2 antibody orantigen-binding fragment thereof into the body of a patient to be testedfor the presence of a tumor associated with TIMP2 expression (e.g.,which expresses TIMP2, for example, on the tumor cell surface) followedby nuclear imaging of the body of the patient to detect the presence ofthe labeled antibody or fragment e.g., at loci comprising a highconcentration of the antibody or fragment which are bound to the tumor.The detection of the loci indicates the presence of the TIMP2⁺ tumor andtumor cells.

Imaging techniques include SPECT imaging (single photon emissioncomputed tomography) or PET imaging (positron emission tomography).Labels include e.g., iodine-123 (¹²³I) and technetium-99m (^(99m)Tc),e.g., in conjunction with SPECT imaging or ¹¹C, ¹³N, ¹⁵O or ¹⁸F, e.g.,in conjunction with PET imaging or Indium-111 (See e.g., Gordon et al.,(2005) International Rev. Neurobiol. 67:385-440).

Pharmaceutical Compositions and Administration

To prepare pharmaceutical or sterile compositions of the anti-TIMP2antibodies and antigen-binding fragments of the invention, the antibodyor antigen-binding fragment thereof is admixed with a pharmaceuticallyacceptable carrier or excipient. See, e.g., Remington's PharmaceuticalSciences and U.S. Pharmacopeia: National Formulary, Mack PublishingCompany, Easton, Pa. (1984).

Formulations of therapeutic and diagnostic agents may be prepared bymixing with acceptable carriers, excipients, or stabilizers in the formof, e.g., lyophilized powders, slurries, aqueous solutions orsuspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's ThePharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;Gennaro (2000) Remington: The Science and Practice of Pharmacy,Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.)(1993) Pharmaceutical Dosage Forms: Parenteral Medications, MarcelDekker, N.Y.; Lieberman, et al. (eds.) (1990) Pharmaceutical DosageForms: Tablets, Marcel Dekker, N.Y.; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, N.Y.;Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, MarcelDekker, Inc., New York, N.Y.).

Toxicity and therapeutic efficacy of the antibodies of the invention,administered alone or in combination with another therapeutic agent, canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index (LD₅₀/ED₅₀). The data obtained fromthese cell culture assays and animal studies can be used in formulatinga range of dosage for use in human. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administration.

In a further embodiment, a further therapeutic agent that isadministered to a subject in association with an anti-TIMP2 antibody orantigen-binding fragment thereof of the invention in accordance with thePhysicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (Nov.1, 2002)).

The mode of administration can vary. Routes of administration includeoral, rectal, transmucosal, intestinal, parenteral; intramuscular,subcutaneous, intradermal, intramedullary, intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, intraocular,inhalation, insufflation, topical, cutaneous, transdermal, intratumoral,or intra-arterial.

In particular embodiments, the anti-TIMP2 antibodies or antigen-bindingfragments thereof of the invention can be administered by an invasiveroute such as by injection. In further embodiments of the invention, ananti-TIMP2 antibody or antigen-binding fragment thereof, orpharmaceutical composition thereof, is administered intravenously,subcutaneously, intramuscularly, intraarterially, intratumorally, or byinhalation, aerosol delivery. Administration by non-invasive routes(e.g., orally; for example, in a pill, capsule or tablet) is also withinthe scope of the present invention.

The present invention provides a vessel (e.g., a plastic or glass vial,e.g., with a cap or a chromatography column, hollow bore needle or asyringe cylinder) comprising any of the antibodies or antigen-bindingfragments of the invention or a pharmaceutical composition thereof. Thepresent invention also provides an injection device comprising any ofthe antibodies or antigen-binding fragments of the invention or apharmaceutical composition thereof. An injection device is a device thatintroduces a substance into the body of a patient via a parenteralroute, e.g., intramuscular, subcutaneous or intravenous. For example, aninjection device may be a syringe (e.g., pre-filled with thepharmaceutical composition, such as an auto-injector) which, forexample, includes a cylinder or barrel for holding fluid to be injected(e.g., antibody or fragment or a pharmaceutical composition thereof), aneedle for piecing skin and/or blood vessels for injection of the fluid;and a plunger for pushing the fluid out of the cylinder and through theneedle bore. In an embodiment of the invention, an injection device thatcomprises an antibody or antigen-binding fragment thereof of the presentinvention or a pharmaceutical composition thereof is an intravenous (IV)injection device. Such a device includes the antibody or fragment or apharmaceutical composition thereof in a cannula or trocar/needle whichmay be attached to a tube which may be attached to a bag or reservoirfor holding fluid (e.g., saline; or lactated ringer solution comprisingNaCl, sodium lactate, KCl, CaCl₂ and optionally including glucose)introduced into the body of the patient through the cannula ortrocar/needle. The antibody or fragment or a pharmaceutical compositionthereof may, in an embodiment of the invention, be introduced into thedevice once the trocar and cannula are inserted into the vein of asubject and the trocar is removed from the inserted cannula. The IVdevice may, for example, be inserted into a peripheral vein (e.g., inthe hand or arm); the superior vena cava or inferior vena cava, orwithin the right atrium of the heart (e.g., a central IV); or into asubclavian, internal jugular, or a femoral vein and, for example,advanced toward the heart until it reaches the superior vena cava orright atrium (e.g., a central venous line). In an embodiment of theinvention, an injection device is an autoinjector; a jet injector or anexternal infusion pump. A jet injector uses a high-pressure narrow jetof liquid which penetrate the epidermis to introduce the antibody orfragment or a pharmaceutical composition thereof to a patient's body.External infusion pumps are medical devices that deliver the antibody orfragment or a pharmaceutical composition thereof into a patient's bodyin controlled amounts. External infusion pumps may be poweredelectrically or mechanically. Different pumps operate in different ways,for example, a syringe pump holds fluid in the reservoir of a syringe,and a moveable piston controls fluid delivery, an elastomeric pump holdsfluid in a stretchable balloon reservoir, and pressure from the elasticwalls of the balloon drives fluid delivery. In a peristaltic pump, a setof rollers pinches down on a length of flexible tubing, pushing fluidforward. In a multi-channel pump, fluids can be delivered from multiplereservoirs at multiple rates.

The pharmaceutical compositions disclosed herein may also beadministered with a needleless hypodermic injection device; such as thedevices disclosed in U.S. Pat. Nos. 6,620,135; 6,096,002; 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556. Suchneedleless devices comprising the pharmaceutical composition are alsopart of the present invention. The pharmaceutical compositions disclosedherein may also be administered by infusion. Examples of well-knownimplants and modules for administering the pharmaceutical compositionsinclude those disclosed in: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,447,233, which discloses a medicationinfusion pump for delivering medication at a precise infusion rate; U.S.Pat. No. 4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments. Many other such implants, delivery systems, and modulesare well known to those skilled in the art and those comprising thepharmaceutical compositions of the present invention are within thescope of the present invention.

Alternately, one may administer the anti-TIMP2 antibody orantigen-binding fragment of the invention in a local rather thansystemic manner, for example, via injection of the antibody or fragmentdirectly into a tumor, e.g., a TIMP2⁺ tumor. Furthermore, one mayadminister the antibody or fragment in a targeted drug delivery system,for example, in a liposome coated with a tissue-specific antibody,targeting, for example, a tumor e.g., a TIMP2⁺ tumor, e.g.,characterized by immunopathology. The liposomes will be targeted to andtaken up selectively by the afflicted tissue. Such methods and liposomesare part of the present invention.

The administration regimen depends on several factors, including theserum or tissue turnover rate of the therapeutic antibody orantigen-binding fragment, the level of symptoms, the immunogenicity ofthe therapeutic antibody, and the accessibility of the target cells inthe biological matrix. Preferably, the administration regimen deliverssufficient therapeutic antibody or fragment to effect improvement in thetarget disease state, while simultaneously minimizing undesired sideeffects. Accordingly, the amount of biologic delivered depends in parton the particular therapeutic antibody and the severity of the conditionbeing treated. Guidance in selecting appropriate doses of therapeuticantibodies or fragments is available (see, e.g., Wawrzynczak (1996)Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina(ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, MarcelDekker, New York, N.Y.; Bach (ed.) (1993) Monoclonal Antibodies andPeptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.;Baert, et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al.(1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl.J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med.342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky etal. (2000) New Engl. J. Med. 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment. Generally, the dose begins with an amount somewhat less thanthe optimum dose and it is increased by small increments thereafteruntil the desired or optimum effect is achieved relative to any negativeside effects. Important diagnostic measures include those of symptomsof, e.g., the inflammation or level of inflammatory cytokines produced.In general, it is desirable that a biologic that will be used is derivedfrom the same species as the animal targeted for treatment, therebyminimizing any immune response to the reagent. In the case of humansubjects, for example, humanized and fully human antibodies are may bedesirable.

Antibodies or antigen-binding fragments thereof disclosed herein may beprovided by continuous infusion, or by doses administered, e.g., daily,1-7 times per week, weekly, bi-weekly, monthly, bimonthly, quarterly,semiannually, annually etc. Doses may be provided, e.g., intravenously,subcutaneously, topically, orally, nasally, rectally, intramuscular,intracerebrally, intraspinally, or by inhalation. A total weekly dose isgenerally at least 0.05 μg/kg body weight, more generally at least 0.2μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.25 mg/kg, 1.0 mg/kg,2.0 mg/kg, 5.0 mg/ml, 10 mg/kg, 25 mg/kg, 50 mg/kg or more (see, e.g.,Yang, et al. (2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002)New Engl. J. Med. 346:1692-1698; Liu, et al. (1999) J. Neurol.Neurosurg. Psych. 67:451-456; Portielji, et al. (20003) Cancer Immunol.Immunother. 52:151-144). Doses may also be provided to achieve apre-determined target concentration of anti-TIMP2 antibody in thesubject's serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg/ml or more.In other embodiments, An anti-TIMP2 antibody of the present invention isadministered, e.g., subcutaneously or intravenously, on a weekly,biweekly, “every 4 weeks,” monthly, bimonthly, or quarterly basis at 10,20, 50, 80, 100, 200, 500, 1000 or 2500 mg/subject.

As used herein, the term “effective amount” refer to an amount of ananti-TIMP2 or antigen-binding fragment thereof of the invention that,when administered alone or in combination with an additional therapeuticagent to a cell, tissue, or subject, is effective to cause a measurableimprovement in one or more symptoms of disease, for example cancer orthe progression of cancer. An effective dose further refers to thatamount of the antibody or fragment sufficient to result in at leastpartial amelioration of symptoms, e.g., tumor shrinkage or elimination,lack of tumor growth, increased survival time. When applied to anindividual active ingredient administered alone, an effective doserefers to that ingredient alone. When applied to a combination, aneffective dose refers to combined amounts of the active ingredients thatresult in the therapeutic effect, whether administered in combination,serially or simultaneously. An effective amount of a therapeutic willresult in an improvement of a diagnostic measure or parameter by atleast 10%; usually by at least 20%; preferably at least about 30%; morepreferably at least 40%, and most preferably by at least 50%. Aneffective amount can also result in an improvement in a subjectivemeasure in cases where subjective measures are used to assess diseaseseverity.

Kits

Further provided are kits comprising one or more components thatinclude, but are not limited to, an anti-TIMP2 antibody orantigen-binding fragment, as discussed herein in association with one ormore additional components including, but not limited to apharmaceutically acceptable carrier and/or a therapeutic agent, asdiscussed herein. The antibody or fragment and/or the therapeutic agentcan be formulated as a pure composition or in combination with apharmaceutically acceptable carrier, in a pharmaceutical composition.

In one embodiment, the kit includes an anti-TIMP2 antibody orantigen-binding fragment thereof of the invention or a pharmaceuticalcomposition thereof in one container (e.g., in a sterile glass orplastic vial) and a pharmaceutical composition thereof and/or atherapeutic agent in another container (e.g., in a sterile glass orplastic vial).

In another embodiment, the kit comprises a combination of the invention,including an anti-TIMP2 antibody or antigen-binding fragment thereof ofthe invention along with a pharmaceutically acceptable carrier,optionally in combination with one or more therapeutic agents formulatedtogether, optionally, in a pharmaceutical composition, in a single,common container.

If the kit includes a pharmaceutical composition for parenteraladministration to a subject, the kit can include a device for performingsuch administration. For example, the kit can include one or morehypodermic needles or other injection devices as discussed above.

The kit can include a package insert including information concerningthe pharmaceutical compositions and dosage forms in the kit. Generally,such information aids patients and physicians in using the enclosedpharmaceutical compositions and dosage forms effectively and safely. Forexample, the following information regarding a combination of theinvention may be supplied in the insert: pharmacokinetics,pharmacodynamics, clinical studies, efficacy parameters, indications andusage, contraindications, warnings, precautions, adverse reactions,overdosage, proper dosage and administration, how supplied, properstorage conditions, references, manufacturer/distributor information andpatent information.

As a matter of convenience, an anti-TIMP2 antibody or antigen-bindingfragment thereof of the invention can be provided in a kit, i.e., apackaged combination of reagents in predetermined amounts withinstructions for performing the diagnostic or detection assay. Where theantibody or fragment is labeled with an enzyme, the kit will includesubstrates and cofactors required by the enzyme (e.g., a substrateprecursor which provides the detectable chromophore or fluorophore). Inaddition, other additives may be included such as stabilizers, buffers(e.g., a block buffer or lysis buffer) and the like. The relativeamounts of the various reagents may be varied widely to provide forconcentrations in solution of the reagents which substantially optimizethe sensitivity of the assay. Particularly, the reagents may be providedas dry powders, usually lyophilized, including excipients which ondissolution will provide a reagent solution having the appropriateconcentration.

Also provided are diagnostic or detection reagents and kits comprisingone or more such reagents for use in a variety of detection assays,including for example, immunoassays such as ELISA (sandwich-type orcompetitive format). The kit's components may be pre-attached to a solidsupport, or may be applied to the surface of a solid support when thekit is used. In some embodiments of the invention, the signal generatingmeans may come pre-associated with an antibody or fragment of theinvention or may require combination with one or more components, e.g.,buffers, antibody-enzyme conjugates, enzyme substrates, or the like,prior to use. Kits may also include additional reagents, e.g., blockingreagents for reducing nonspecific binding to the solid phase surface,washing reagents, enzyme substrates, and the like. The solid phasesurface may be in the form of a tube, a bead, a microtiter plate, amicrosphere, or other materials suitable for immobilizing proteins,peptides, or polypeptides. In particular aspects, an enzyme thatcatalyzes the formation of a chemilluminescent or chromogenic product orthe reduction of a chemilluminescent or chromogenic substrate is acomponent of the signal generating means. Such enzymes are well known inthe art. Kits may comprise any of the capture agents and detectionreagents described herein. Optionally the kit may also compriseinstructions for carrying out the methods of the invention.

Also provided is a kit comprising an anti-TIMP2 antibody (e.g.,humanized antibody) or antigen-binding fragment thereof packaged in acontainer, such as a vial or bottle, and further comprising a labelattached to or packaged with the container, the label describing thecontents of the container and providing indications and/or instructionsregarding use of the contents of the container to treat one or moredisease states as described herein.

In one aspect, the kit is for treating cancer and comprises ananti-TIMP2 antibody (e.g., humanized antibody) or antigen-bindingfragment thereof and a further therapeutic agent or a vaccine. The kitmay optionally further include a syringe for parenteral, e.g.,intravenous, administration. In another aspect, the kit comprises ananti-TIMP2 antibody (e.g., humanized antibody) or antigen-bindingfragment thereof and a label attached to or packaged with the containerdescribing use of the antibody or fragment with the vaccine or furthertherapeutic agent. In yet another aspect, the kit comprises the vaccineor further therapeutic agent and a label attached to or packaged withthe container describing use of the vaccine or further therapeutic agentwith the anti-TIMP2 antibody or fragment. In certain embodiments, ananti-TIMP2 antibody and vaccine or further therapeutic agent are inseparate vials or are combined together in the same pharmaceuticalcomposition.

As discussed above in the combination therapy section, concurrentadministration of two therapeutic agents does not require that theagents be administered at the same time or by the same route, as long asthere is an overlap in the time period during which the agents areexerting their therapeutic effect. Simultaneous or sequentialadministration is contemplated, as is administration on different daysor weeks.

The therapeutic and detection kits disclosed herein may also be preparedthat comprise at least one of the antibody, peptide, antigen-bindingfragment, or polynucleotide disclosed herein and instructions for usingthe composition as a detection reagent or therapeutic agent. Containersfor use in such kits may typically comprise at least one vial, testtube, flask, bottle, syringe or other suitable container, into which oneor more of the detection and/or therapeutic composition(s) may beplaced, and preferably suitably aliquoted. Where a second therapeuticagent is also provided, the kit may also contain a second distinctcontainer into which this second detection and/or therapeuticcomposition may be placed. Alternatively, a plurality of compounds maybe prepared in a single pharmaceutical composition, and may be packagedin a single container means, such as a vial, flask, syringe, bottle, orother suitable single container. The kits disclosed herein will alsotypically include a means for containing the vial(s) in closeconfinement for commercial sale, such as, e.g., injection or blow-moldedplastic containers into which the desired vial(s) are retained. Where aradiolabel, chromogenic, fluorigenic, or other type of detectable labelor detecting means is included within the kit, the labeling agent may beprovided either in the same container as the detection or therapeuticcomposition itself, or may alternatively be placed in a second distinctcontainer means into which this second composition may be placed andsuitably aliquoted. Alternatively, the detection reagent and the labelmay be prepared in a single container means, and in most cases, the kitwill also typically include a means for containing the vial(s) in closeconfinement for commercial sale and/or convenient packaging anddelivery.

A device or apparatus for carrying out the detection or monitoringmethods described herein is also provided. Such an apparatus may includea chamber or tube into which sample can be input, a fluid handlingsystem optionally including valves or pumps to direct flow of the samplethrough the device, optionally filters to separate plasma or serum fromblood, mixing chambers for the addition of capture agents or detectionreagents, and optionally a detection device for detecting the amount ofdetectable label bound to the capture agent immunocomplex. The flow ofsample may be passive (e.g., by capillary, hydrostatic, or other forcesthat do not require further manipulation of the device once sample isapplied) or active (e.g., by application of force generated viamechanical pumps, electroosmotic pumps, centrifugal force, or increasedair pressure), or by a combination of active and passive forces.

In further embodiments, also provided is a processor, a computerreadable memory, and a routine stored on the computer readable memoryand adapted to be executed on the processor to perform any of themethods described herein. Examples of suitable computing systems,environments, and/or configurations include personal computers, servercomputers, hand-held or laptop devices, multiprocessor systems,microprocessor-based systems, set top boxes, programmable consumerelectronics, network PCs, minicomputers, mainframe computers,distributed computing environments that include any of the above systemsor devices, or any other systems known in the art.

General Methods

Standard methods in molecular biology are described Sambrook, Fritschand Maniatis (1982 & 1989 2^(nd) Edition, 2001 3^(rd) Edition) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Sambrook and Russell (2001) Molecular Cloning,3^(rd) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego,Calif.). Standard methods also appear in Ausbel, et al. (2001) CurrentProtocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. NewYork, N.Y., which describes cloning in bacterial cells and DNAmutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2),glycoconjugates and protein expression (Vol. 3), and bioinformatics(Vol. 4).

Methods for protein purification including immunoprecipitation,chromatography, electrophoresis, centrifugation, and crystallization aredescribed (Coligan, et al. (2000) Current Protocols in Protein Science,Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis,chemical modification, post-translational modification, production offusion proteins, glycosylation of proteins are described (see, e.g.,Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2,John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) CurrentProtocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY,N.Y., pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for LifeScience Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech(2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production,purification, and fragmentation of polyclonal and monoclonal antibodiesare described (Coligan, et al. (2001) Current Protcols in Immunology,Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999)Using Antibodies, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Harlow and Lane, supra). Standard techniques forcharacterizing ligand/receptor interactions are available (see, e.g.,Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, JohnWiley, Inc., New York).

Monoclonal, polyclonal, and humanized antibodies can be prepared (see,e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ.Press, New York, N.Y.; Kontermann and Dubel (eds.) (2001) AntibodyEngineering, Springer-Verlag, N.Y.; Harlow and Lane (1988) Antibodies ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., pp. 139-243; Carpenter, et al. (2000) J. Immunol.165:6205; He, et al. (1998) J. Immunol. 160:1029; Tang et al. (1999) J.Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem.272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote andWinter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

An alternative to humanization is to use human antibody librariesdisplayed on phage or human antibody libraries in transgenic mice(Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995)Nature Medicine 1:837-839; Mendez et al. (1997) Nature Genetics15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377;Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; Kay et al. (1996)Phage Display of Peptides and Proteins: A Laboratory Manual, AcademicPress, San Diego, Calif.; de Bruin et al. (1999) Nature Biotechnol.17:397-399).

Single chain antibodies and diabodies are described (see, e.g., Maleckiet al. (2002) Proc. Natl. Acad. Sci. USA 99:213-218; Conrath et al.(2001) J. Biol. Chem. 276:7346-7350; Desmyter et al. (2001) J. Biol.Chem. 276:26285-26290; Hudson and Kortt (1999) J. Immunol. Methods231:177-189; and U.S. Pat. No. 4,946,778). Bifunctional antibodies areprovided (see, e.g., Mack, et al. (1995) Proc. Natl. Acad. Sci. USA92:7021-7025; Carter (2001) J. Immunol. Methods 248:7-15; Volkel, et al.(2001) Protein Engineering 14:815-823; Segal, et al. (2001) J. Immunol.Methods 248:1-6; Brennan, et al. (1985) Science 229:81-83; Raso, et al.(1997) J. Biol. Chem. 272:27623; Morrison (1985) Science 229:1202-1207;Traunecker, et al. (1991) EMBO J. 10:3655-3659; and U.S. Pat. Nos.5,932,448, 5,532,210, and 6,129,914).

Multispecific antibodies are also provided (see, e.g., Azzoni et al.(1998) J. Immunol. 161:3493; Kita et al. (1999) J. Immunol. 162:6901;Merchant et al. (2000) J. Biol. Chem. 74:9115; Pandey et al. (2000) J.Biol. Chem. 275:38633; Zheng et al. (2001) J. Biol Chem. 276:12999;Propst et al. (2000) J. Immunol. 165:2214; Long (1999) Ann. Rev.Immunol. 17:875); Labrijin et al., Proc. Natl. Acad. Sci. USA 110:5145-50, 2013; de Jong et al., PLOS Biol 14(1): e1002344, 2016(doi:10.1371/journal.pbio.1002344).

Purification of antigen is not necessary for the generation ofantibodies. Animals can be immunized with cells bearing the antigen ofinterest. Splenocytes can then be isolated from the immunized animals,and the splenocytes can fused with a myeloma cell line to produce ahybridoma (see, e.g., Meyaard et al. (1997) Immunity 7:283-290; Wrightet al. (2000) Immunity 13:233-242; Preston et al., supra; Kaithamana etal. (1999) J. Immunol. 163:5157-5164).

Antibodies can be conjugated, e.g., to small drug molecules, enzymes,liposomes, polyethylene glycol (PEG). Antibodies are useful fortherapeutic, diagnostic, kit or other purposes, and include antibodiescoupled, e.g., to dyes, radioisotopes, enzymes, or metals, e.g.,colloidal gold (see, e.g., Le Doussal et al. (1991) J. Immunol.146:169-175; Gibellini et al. (1998) J. Immunol. 160:3891-3898; Hsingand Bishop (1999) J. Immunol. 162:2804-2811; Everts et al. (2002) J.Immunol. 168:883-889).

Methods for flow cytometry, including fluorescence activated cellsorting (FACS), are available (see, e.g., Owens, et al. (1994) FlowCytometry Principles for Clinical Laboratory Practice, John Wiley andSons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2^(nd) ed.;Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry, JohnWiley and Sons, Hoboken, N.J.). Fluorescent reagents suitable formodifying nucleic acids, including nucleic acid primers and probes,polypeptides, and antibodies, for use, e.g., as diagnostic reagents, areavailable (Molecular Probes (2003) Catalogue, Molecular Probes, Inc.,Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

Standard methods of histology of the immune system are described (see,e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology andPathology, Springer Verlag, New York, N.Y.; Hiatt, et al. (2000) ColorAtlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.;Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, NewYork, N.Y.).

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains,glycosylation sites, and sequence alignments, are available (see, e.g.,GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, Md.); GCG WisconsinPackage (Accelrys, Inc., San Diego, Calif.); DeCypher® (TimeLogic Corp.,Crystal Bay, Nev.); Menne, et al. (2000) Bioinformatics 16: 741-742;Menne, et al. (2000) Bioinformatics Applications Note 16:741-742; Wren,et al. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res.14:4683-4690).

EXAMPLES Example 1 Monoclonal Antibody Development in Rabbits

Female New Zealand Rabbits were immunized by subcutaneous injections(SQ) with antigen/adjuvant emulsions. Primary immunization was done withComplete Freund's Adjuvant and Incomplete Freund's Adjuvant was used forall subsequent boosts. Rabbits were injected SQ every three weeks at 250μg TIMP2 antigen per rabbit (alternating two sites, hips and scapulas).A test bleed was taken from the marginal ear vein seven days after thesecond boost. This test bleed (immune sera) was tested by indirect ELISAassay to determine if immune response of the rabbit was adequate formonoclonal antibody development. The best responding rabbit was given afinal SQ boost and four days later was euthanized via exsanguination.The whole blood was collected via cardiac puncture. B cells producingantibody of interest were identified by indirect ELISA on target antigenand immunoglobulin genes were isolated. Heavy and light chains werecloned into separate mammalian expression vectors, transfected into HEKcells (transient transfection), and tissue culture supernatantcontaining rabbit monoclonal antibodies were harvested.

An antibody designated 40H2-40K3 was isolated, and the nucleic acid andprotein sequences determined as follows:

Heavy chain SEQ ID NO: 9atggagactgggctgcgctggcttctcctggtcgctgtgctcaaaggtgtccagtgtcagtcgttggaggagtccgggggagacctggtcaagcctgagggatccctgacactcacctgcacagcctctggattcaccatcagttctaactactacatgtgctgggtccgccaggctccagggaaggggctggagtgggtcgcatgcattttgggtggtagtggtacttacacttactacgcgacctgggcgaaaggccgattcaccatctccaaaacctcgtcgaccacggtgactctgcaaatgcccagtctgacagccgcggacacggccacctatttctgtgcgagacaggcccctgccgatacttatctttatggtggttatggtccctttaacttgtggggccaagggaccctcgtcaccgtctcgagcggacagccgaaagccccgtcggtgtttccactggcgccctgctgtggcgatacgccttcgtccaccgtgacccttggctgtcttgtgaagggttaccttcccgagccggtcactgtaacatggaattcagggacactcacgaacggggtcaggactttcccatcagtcagacagtcatccggtctgtattcacttagctcggtagtgtccgtaacttcctccagccagccggtaacatgtaacgtagcgcaccccgccaccaataccaaggtggacaagaccgtggccccctcaacatgctcgaaacctacgtgccctccgcctgaacttctcgggggtcccagcgtctttatcttccctcctaagcccaaagatacgctgatgatctcgcggaccccggaggtgacttgtgtcgtggtcgatgtctcccaagacgatcccgaagtacagttcacctggtacattaacaacgagcaagtccgaacggccaggccaccgttgcgcgagcagcaattcaattcgacgatccgggtggtatcaacgttgccgatcactcatcaggactggttgc A agggaaggaattcaaatgcaaggtccacaacaaggcccttccggcaccaatcgagaaaacgatcagcaaggcgagggggcagcccctggaacccaaggtctatacaatgggaccacccagggaagagttgtcatcccggtccgtatcgcttacatgcatgattaacggtttctatccttcagacatttcagtagagtgggagaagaatggaaaagccgaagataactacaaaacaacccccgcagtacttgactccgacggatcgtacttcttgtacaacaagctctcggtgcccacgtcagaatggcaacgaggggatgtctttacatgctcggtgatgcatgaggcactccacaatcattacacgcagaaaagcatctcccgctcgccgggaaagtgatag SEQ ID NO: 10:metglrwlllvavlkgvqcqsleesggdlvkpegsltltctasgftissnyymcwvrqapgkglewvacilggsgtytyyatwakgrftisktssttvtlqmpsltaadtatyfcarqapadtylyggygpfnlwgqgtlvtvssgqpkapsvfplapccgdtpsstvtlgclvkgylpepvtvtwnsgtltngvrtfpsvrqssglyslssvvsvtsssqpvtcnvahpatntkvdktvapstcskptcpppellggpsvfifppkpkdtlmisrtpevtcvvvdvsqddpevqftwyinneqvrtarpplreqqfnstirvvstlpithqdwl Q gkefkckvhnkalpapiektiskargqplepkvytmgppreelssrsysltcmingfypsdisvewekngkaednykttpavldsdgsyflynklsvptsewqrgdvftcsvmhealhnhytqksisrspgk*

The nucleotide and corresponding constant domain amino acid marked asbold and underlined may be replaced, e.g., G>A resulting in amino-acidsubstitution e.g., R>Q. Preferred residues at this position: R, Q, N.

FR1: (SEQ ID NO: 11) QSLEESGGDLVKPEGSLTLTCTAS CDR1: (SEQ ID NO: 4)GFTISSNYY FR2: (SEQ ID NO: 12) MCWVRQAPGKGLEWVAC CDR2: (SEQ ID NO: 5)ILGGSGTYT FR3: (SEQ ID NO: 13) YYATWAKGRFTISKTSSTTVTLQMPSLTAADTATYFCCDR3: (SEQ ID NO: 6) ARQAPADTYLYGGYGPFNL FR4: (SEQ ID NO: 14)WGQGTLVTVSS Light chain SEQ ID NO: 15:atggacacgagggcccccactcagctgctggggctcctgctgctctggctcccaggtgccagatgtgccgacatcgtgatgacccagactccatcctccgtggaggcagctgtgggaggcacagtcaccatcaagtgccaggccagtgagagcattagcggttggttggcctggtatcagcagaaaccagggcagcctcccaagctcctgatctacagggcatccactctggaatctggggtcccatcgcggttcaaaggcagtggatctgggacagagttcactctcaccatcagcgacctggagtgtgccgatgctgccacttattattgtcaatgcagttatggtattaatggtaatagtgagcatggtaatcctttcggcggagggaccgaggtggtggtcaaacgtacgcccgtggcacccactgtactcctgtttccgccttcctcggatgaggtggcgacgggcacggtcacaatcgtctgcgtggcgaataagtactttccggatgtcacagtgacgtgggaggtggacgggacaacacagaccacaggtattgaaaacagcaaaacaccgcagaattcggctgactgtacgtataacttgtcctccactcttacgttgacatcaacacagtacaattcgcacaaggagtatacgtgcaaggtaacccagggtacgacaagcgtagtccagtccttcagcaggaagaactgctgataa SEQ ID NO: 16mdtraptqllgllllw1pgarcadivmtqtpssveaavggtvtikcciasesisgwlawyqqkpgqppklliyrastlesgvpsrfkgsgsgteftltisdlecadaatyycqcsygingnsehgnpfgggtevvvkrtpvaptvllfppssdevatgtvtivcvankyfpdvtvtwevdgttqttgiensktpqnsadctynlsstltltstqynshkeytckvtqgttsvvqsfsrknc FR1: (SEQ ID NO: 17)DIVMTQTPSSVEAAVGGTVTIKCQAS CDR1: (SEQ ID NO: 1) ESISGW FR2:(SEQ ID NO: 18) LAWYQQKPGQPPKLLIY CDR2: (SEQ ID NO: 2) RAS FR3:(SEQ ID NO: 19) TLESGVPSRFKGSGSGTEFTLTISDLECADAATYYC CDR3:(SEQ ID NO: 3) QCSYGINGNSEHGNP FR4: (SEQ ID NO: 20) FGGGTEVVVK

An antibody designated 6E2.1 was isolated, and the nucleic acid andprotein sequences determined as follows:

Heavy chain SEQ ID NO 32:atgggctggagctgcattattctgtttctggtgagcaccgcgaccggcgtgcatagccaggtgcagctgcagcagagcggcccgcagctggtgcgcccgggcgcgagcgtgaaaattagctgcaaagcgagcggctatagctttaccagctattggatgcattgggtgaaacagcgcccgggccagggcctggaatggattggcgtgattgatccgagcgatagcgaaacccgcctgaaccagaaatttaaagataaagcgaccctgaccgtggataaaagcagcagcaccgcgtatatgcagctgaacagcccgaccagcgaagatagcgcggtgtattattgcgcgcgccgcgattatggcagccgctatgatgcgatggattattggggccagggcaccagcgtgaccgtgagcagcgcgaaaaccaccccgccgagcgtgtatccgctggcgccgggcagcgcggcgcagaccaacagcatggtgaccctgggctgcctggtgaaaggctattttccggaaccggtgaccgtgacctggaac SEQ ID NO: 33:MGWSCIILFLVSTATGVHSQVQLQQSGPQLVRPGASVKISCKASGYSFTSYWMHWVKQRPGQGLEWIGVIDPSDSETRLNQKFKDKATLTVDKSSSTAYMQLNSPTSEDSAVYYCARRDYGSRYDAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWN* FR1: (SEQ ID NO: 34)QSGPQLVRPGASVKISCKAS CDR1: (SEQ ID NO: 27) GYSFTSYW FR2: (SEQ ID NO: 35)MHWVKQRPGQGLEWIGV CDR2: (SEQ ID NO: 28) IDPSDSET FR3: (SEQ ID NO: 36)RLNQKFKDKATLTVDKSSSTAYMQLNSPTSEDSAVYYC CDR3: (SEQ ID NO: 29)ARRDYGSRYDAMDY FR4: (SEQ ID NO: 37) WGQGTSVTVSS Light chainSEQ ID NO: 38: atgaaatttccgagccagctgctgctgtttctgctgtttcgcattaccggcattatttgcgatattcagatgacccagagcagcagctatctgagcgtgagcctgggcggccgcgtgaccattacctgcaaagcgagcgatcatattaacaactggctggcgtggtatcagcagaaaccgggcaacgcgccgcgcctgctgattagcggcgcgaccagcctggaaaccggcgtgccgagccgctttagcggcagcggcagcggcaaagattataccctgagcattaccagcctgcagaccgaagatgtggcgacctattattgccagcagtattggagcaccccgtttacctttggcagcggcaccaaactggaaattaaacgcgcggatgcggcgccgaccgtgagcatttttccgccgagcagcgaacagctgagcaac SEQ ID NO: 39MKFPSQLLLFLLFRITGIICDIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLSN FR1: (SEQ ID NO: 40)DIQMTQSSSYLSVSLGGRVTITCKAS CDR1: (SEQ ID NO: 24) DHINNW FR2:(SEQ ID NO: 41) LAWYQQKPGNAPRLLI CDR2: (SEQ ID NO: 25) SGA FR3:(SEQ ID NO: 42) TSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYC CDR3:(SEQ ID NO: 26) QQYWSTPFT FR4: (SEQ ID NO: 43) FGSGTKLEIK

Example 2 Screening of Antibodies with Patient Samples (Microtiter-BasedELISA Method)

Materials:

96-well high bind ELISA plates-Costar 3590 (Corning)

ELISA coating buffer: PBS

ELISA wash buffer: PBS with 0.02% Tween-20

ELISA blocking Buffer (Thermo Pierce, catalogue number N502)

ELISA reagent diluent: 200 mM Tris, 1% BSA (BioFx), 0.05% Tween-20, pH8.1

Neutravidin-HRP conjugate (Thermo Pierce, catalogue number 31001)

1-Step Ultra TMB substrate (R&D systems, catalogue number 34028)

Stop solution: 2N sulfuric acid

Capture antibodies (6E2.1)

Biotin conjugated detection antibodies (40H2-40K3)

Recombinant human TIMP2 (Peprotech, catalogue number 410-02)

EXLx405 plate washer (Biotek)

Multiskan FC plate reader (Fisher Scientific)

Testing Procedure

Purified, recombinant TIMP2 analyte was spiked into Reagent Diluent andserially diluted to generate a set of standard samples covering a rangeof concentrations. Frozen single-use aliquots of patient samples werethawed in a room temperature water bath for 10 minutes, and then dilutedto desired level with Reagent Diluent.

100 μL of 5 μg/mL Capture Antibody solution prepared in coating bufferwas added to each well on a 96-well high bind ELISA plate and incubatedover night at room temperature (22° C. to 25° C.). Each well wasaspirated and washed three times with 300 μL of wash buffer using anautowasher. Then 250 μL of ELISA blocking buffer was added to each well.After an incubation of 2 hours at room temperature, the aspiration/washstep described above was repeated.

100 μL of standard or patient samples was added to each well of theprepared plate and incubated at room temperature on a horizontal orbitalshaker. After 2 hours of incubation, the plate was washed as describedabove. Then 100 μL of 0.1 μg/mL detection antibody solution prepared inreagent diluent was added to each well. After incubation for 1 hour atroom temperature, the plate was washed again. A 0.1 μg/mL solution ofneutravidin-HRP conjugate was prepared in reagent diluent, and 100 μL ofthis solution was added to each well. The plate was incubated for 1 hourat room temperature and washed. 100 μL of 1-step ultra TMB substrate wasadded to each well, incubated at room temperature for 10 minutesprotected from light, followed by 50 μL of stop solution. The opticaldensity in each well was measured with a microplate reader set to awavelength of 450 nm.

Example 3 Screening of Antibodies with Patient Samples (Lateral FlowStrip Testing Method)

Materials:

Nitrocellulose membrane

Backing card

Sample pad

Wicking pad

Membrane blocking buffer: 10 mM Sodium phosphate, 0.1% sucrose, 0.1%BSA, 0.2% PVP-40, pH 8.0

Sample pad blocking buffer: 5 mM Borate, 0.1% Tween-20, 0.25% PVP-40,0.5% BSA, pH 8.5

Running buffer J: 500 mM Tris, 0.2% 10G, 0.35% Tween-20, 0.25% PVP-40,pH 8.5

Fluorescently-conjugated antibodies (40H2-40K3)

Test line antibodies (6E2.1)

Goat-anti-mouse positive control antibodies

Recombinant human TIMP-2

Strip Assembly

Nitrocellulose membranes were striped with test line antibodies using anAD3050 aspirate dispense system, blocked with the membrane blockingbuffer and dried at 37° C. for 30 min. After curing over night in adesiccator, the striped and blocked nitrocellulose membranes werelaminated onto backing cards with wicking pads and sample padspre-treated with the sample pad blocking buffer. The cards were cut into5 mm wide test strips, which were then placed into cartridges.

Sample Preparation

Purified, recombinant TIMP-2 analyte was spiked into Running buffer Jand serially diluted to generate a set of standard samples covering arange of concentrations. Frozen single-use aliquots of patient sampleswere thawed in a room temperature water bath for 10 minutes, and thendiluted to desired level with Running buffer J.

Testing Procedure

10 μL of fluorescently conjugated antibody (0.025 μg/μL) in PBS wasadded to 100 μL of sample. 100 μL of this solution was then loaded intothe input port on the cartridge. Results was read at t=20 minutes usinga fluorescence reader and associated software.

Example 4 Epitope Mapping Using Pepscan Method Synthesis of Peptides andPepscan Screening

Synthetic linear peptides were synthesized and screened as described bySlootstra et al. (Slootstra et al., 1996, Mol. Diversity 1, 87-96) andTimmerman et al. (Timmerman et al., 2007, J, Mol Recognit 20, 283-299).The binding of antibody to each peptide was tested in a PEPSCAN-basedenzyme-linked immuno assay (ELISA). The covalently linked peptides wereincubated with sample (for example 1 μg/mL antibody diluted in a PBSsolution containing 5% horse serum (vol/vol) and 4% ovalbumin(weight/vol)) and 1% Tween 80 at 4° C. overnight. After washing, thepeptides were incubated with Goat-anti-Rabbit HRP (JacksonImmunoresearch) for 1 hour at 25° C., and subsequently, after washingthe peroxidase substrate 2,2′-azino-di-3-ethylbenzthiazoline sulfonate(ABTS) and 2 μg/mL 3% H₂O₂ were added. After 1 hour the colordevelopment was measured.

Peptide set 1 consisted of a nested linear 15-mer peptides from theTIMP-2 sequence having a step of 1 amino acid residue, while peptide set2 consisted of an identical set of peptides with a double alanine mutantat positions 11 and 12 of the peptide. By way of example, three peptidesin peptide set 1, starting from the first residue of native TIMP2 (SEQID NO: 45) would be:

(SEQ ID NO: 46) MGAAARTLRLALGLL (SEQ ID NO: 47) GAAARTLRLALGLLL(SEQ ID NO: 48) AAARTLRLALGLLLLWith the corresponding mutated sequences being:

(SEQ ID NO: 49) MGAAARTLRLGAGLL (SEQ ID NO: 50) GAAARTLRLAAALLL(SEQ ID NO: 51) AAARTLRLALAALLLNote that when an alanine is present in the native sequence, it isreplaced in the corresponding mutated sequence with a glycine. Peptidesthat show high binding on Set 1 but not on Set 2 identify criticalresidues for binding.

Using this methodology, the dominant epitope for the antibody 6E2.1 wasidentified as ₁₃₂PWDTLSTTQKK₁₄₂ (SEQ ID NO: 44), with the underlinedresidues being the most critical residues.

Antibody 40H2-40K3 exhibited three binding regions ₁₄₅NHRYQMGCECKI₁₅₆(SEQ ID NO: 21), ₃₃HPQQAFCNA₄₁ (SEQ ID NO: 22), and ₁₉₆RSDGSCAWYR₂₀₅(SEQ ID NO: 23), indicating a likely discontinuous epitope. The corebinding region was identified as ₁₄₅NHRYQMGCECKI₁₅₆ (SEQ ID NO: 21).

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention. The examplesprovided herein are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Modifications therein and other uses will occur to thoseskilled in the art. These modifications are encompassed within thespirit of the invention and are defined by the scope of the claims.

The use of “or” herein means “and/or” unless stated otherwise.Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,”and “including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although any methods andreagents similar or equivalent to those described herein can be used inthe practice of the disclosed methods and compositions, the exemplarymethods and materials are now described.

All publications mentioned herein are incorporated herein by referencein full for the purpose of describing and disclosing the methodologies,which are described in the publications, which might be used inconnection with the description herein. All patents and publicationsmentioned in the specification are indicative of the levels of those ofordinary skill in the art to which the invention pertains prior to thefiling date of the disclosure. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior disclosure.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A monoclonal antibody that binds to human tissueinhibitor of metalloproteinases 2 (TIMP2) protein, wherein the antibodycomprises: (a) a light chain variable region complementarity determiningregion 1 (CDR1) comprising the amino acid sequence ESISGW (SEQ ID NO:1), a light chain variable region CDR2 comprising the amino acidsequence RAS (SEQ ID NO: 2), and a light chain variable region CDR3comprising the amino acid sequence QCSYGINGNSEHGNP (SEQ ID NO: 3); and(b) a heavy chain variable region CDR1 comprising the amino acidsequence GFTISSNYY (SEQ ID NO:4), a heavy chain variable region CDR2comprising the amino acid sequence ILGGSGTYT (SEQ ID NO:5), and a heavychain variable region CDR3 comprising the amino acid sequenceARQAPADTYLYGGYGPFNL (SEQ ID NO:6).
 2. The monoclonal antibody of claim1, wherein the light chain variable region comprises an amino acidsequence at least 90% identical to that set forth as SEQ ID NO: 7 andthe heavy chain variable region comprises an amino acid sequence atleast 90% identical to that set forth as SEQ ID NO:
 8. 3. The monoclonalantibody of claim 2, wherein the light chain variable region comprisesan amino acid sequence set forth as SEQ ID NO: 7 and the heavy chainvariable region comprises an amino acid sequence set forth as SEQ ID NO:8.
 4. The monoclonal antibody of claim 1, wherein the light chaincomprises an amino acid sequence at least 90% identical to that setforth as SEQ ID NO: 16 and the heavy chain comprises an amino acidsequence at least 90% identical to that set forth as SEQ ID NO:
 10. 5.The monoclonal antibody of claim 4, wherein the light chain comprises anamino acid sequence set forth as SEQ ID NO: 16 and the heavy chaincomprises an amino acid sequence set forth as SEQ ID NO:
 10. 6. Themonoclonal antibody of claim 1, wherein the antibody is a rabbitantibody.
 7. A method for determining the presence or amount of humantissue inhibitor of metalloproteinases 2 (TIMP2) protein in a biologicalsample, the method comprising: performing an immunoassay on thebiological sample with first and second monoclonal antibodies whichtogether form sandwich complexes with human TIMP2 protein, wherein theimmunoassay provides a detectable signal that is related to the presenceor amount of human TIMP2 protein in the biological sample bound in thesandwich complexes; and relating the detectable signal to the presenceor amount of human TIMP2 protein in the biological sample, wherein thefirst monoclonal antibody comprises: the monoclonal antibody as in anyof claims 1-6.
 8. The method of claim 7, wherein the minimum detectableconcentration is 10 ng/mL or less.